Fusion protein based on functional chaperonin and electrochemical application thereof

Abstract

The invention belongs to the technical field of enzyme engineering and electrochemical crossing, discloses a fusion protein based on functional chaperonin and electrochemical application of the fusion protein, and particularly discloses the fusion protein which comprises the functional chaperonin and NOx oxidase. The fusion protein comprises NOx oxidase and functional chaperonin, the functional chaperonin serves as an electron transfer mediator between the NOx oxidase and an electrode, the redox potential of the functional chaperonin is low, and therefore the direct electron transfer process between the enzyme and the electrode under the low potential is achieved. According to the method disclosed by the invention, cyclic oxidation regeneration (electrochemical method) of the cofactor FAD / FADH2 and efficient, controllable and continuous oxidation (enzymatic method) of the substrate NADH are realized through an electrochemical-enzyme coupling method, and a basis is provided for multi-enzyme cascade application to preparation and synthesis of high-added-value chemicals.

Description

Technical Field

[0001] The invention belongs to the cross-technical field of enzyme engineering and electrochemistry, and specifically relates to a fusion protein based on a functional chaperone protein and its electrochemical application. Background Art

[0002] Oxidoreductases are a class of enzymes that catalyze redox reactions and achieve their biological functions primarily by transferring electrons from one molecule (donor) to another molecule (acceptor). Oxidoreductases play a key role in many biological processes, including cellular respiration, photosynthesis, detoxification, and other biochemical reactions related to metabolism and energy conversion. Based on their redox potential characteristics, enzyme-catalyzed redox reactions can be applied to biofuel cells, enzyme reactors, and enzyme biosensors. In these devices, the cofactors / coenzymes of the redox enzymes achieve redox regeneration through electron exchange through electron mediators or direct electrodes, and the corresponding transfer processes are defined as indirect electron transfer (MET) and direct electron transfer (DET), respectively. The electron transfer mechanism between the enzyme and the electrode surface can be explained by the Marcus charge transfer theory. The theory states that the electron transfer rate of a redox reaction depends on the change in the Gibbs free energy of the reaction (i.e., the potential difference), the distance between the redox centers, and the reorganization energy. In theory, the maximum effective distance for electron transfer within a protein is When these conditions are favorable, direct electron transfer (DET) can occur between the enzyme and the electrode.

[0003] However, there are many obstacles to achieving direct electrochemistry of redox enzymes. First, direct contact of enzymes with bare metal surfaces usually causes changes in their structure and function, leading to inactivation and inhibiting electron transfer on the electrode. Second, the active center of the enzyme is usually deeply buried inside its structure and difficult to access the electrode surface, so it is extremely difficult to achieve direct electron transfer. However, some enzymes can achieve long-distance electron transfer very well. For example, in polysulfide iron cluster hydrogenase, the cofactor chain can transfer electrons buried in the redox active site to other cofactors, thereby completing electron transfer. In actual operation, in order to achieve effective direct electron transfer (DET), close contact between the distal cofactor in the chain and the electrode is crucial. By specifically immobilizing and modifying the protein or electrode surface, the interface properties can be optimized, the electroactive center can be exposed, and the rapid electron transfer between the protein and the electrode can be promoted. Studies have shown that the use of nanomaterials (such as gold nanoparticles and carbon nanotubes) to modify electrodes can achieve direct electron transfer of proteins such as cytochrome C, glucose oxidase, and hemoglobin. However, these modification methods often require the use of cross-linking agents and repeated washing and purification after modification, which has an adverse effect on protein activity and stability.

[0004] NAD(P)H oxidase (NOx) is a class of enzymes that use NAD(P)H as an electron donor and flavin compounds (such as FAD, FMN, etc.) as cofactors. It uses oxygen as the final electron acceptor, and the byproduct is water or hydrogen peroxide. In the actual reaction system, as the reaction proceeds, the dissolved oxygen content gradually decreases, making it difficult for FAD / FADH2 to recycle and regenerate in time, thus affecting the continuous NADH oxidation process. To solve this problem, stirring or continuous oxygenation is usually used, but these methods often reduce the stability of the enzyme. Therefore, in actual production applications, it is necessary to make a reasonable trade-off between the reduction in enzyme activity caused by insufficient dissolved oxygen and the reduction in enzyme stability caused by external means. Fortunately, when we use other oxidants such as electrochemical anodes as electron acceptors, electrons can eventually flow to the anode, thereby effectively solving the problem of insufficient dissolved oxygen. Similar to other oxidoreductases, the redox center of NOx (FAD / FADH2) is also deeply buried inside its structure, so direct electron transfer between the enzyme and the electrode still faces huge challenges. Summary of the invention

[0005] In view of the electron transfer mediator properties of functional chaperone proteins, they can serve as an 'intermediary' for electron transfer between NOx and electrodes. Heme is its functional unit, in which the iron atom at the center of the porphyrin macroheterocyclic structure can be converted between different valence states, thereby achieving efficient electron transfer. The present invention has developed a direct electron transfer enzyme electrode at low potential based on functional chaperone proteins. The invention provides important technical support for achieving efficient and controllable coenzyme regeneration, and at the same time provides a preliminary foundation for the application of multi-enzyme cascades in the preparation and synthesis of high value-added chemicals.

[0006] The purpose of the first aspect of the present invention is to provide a fusion protein.

[0007] The purpose of the second aspect of the present invention is to provide biological materials related to the fusion protein of the first aspect of the present invention.

[0008] The third aspect of the present invention aims to provide the use of the fusion protein of the first aspect of the present invention or the biomaterial of the second aspect of the present invention in the preparation of an enzyme electrode.

[0009] The fourth aspect of the present invention aims to provide an enzyme electrode.

[0010] The purpose of the fifth aspect of the present invention is to provide a method for preparing the enzyme electrode of the fourth aspect of the present invention.

[0011] The sixth aspect of the present invention aims to provide a bioelectrochemical reactor.

[0012] The seventh aspect of the present invention aims to provide a method for preparing coenzyme NAD+.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] Since the active center of the enzyme is deeply buried inside its structure, it is difficult to establish an effective electron transfer channel between the enzyme and the electrode. The present invention introduces a functional chaperone protein into the NOx oxidase by means of genetic engineering / enzyme engineering, and uses the chaperone protein as an "endogenous" electron transfer carrier. Based on this, an electrochemical-enzyme coupling method based on the functional chaperone protein is developed to achieve direct electron transfer of NOx at low potential.

[0015] The first aspect of the present invention provides a fusion protein comprising a functional chaperone protein and NOx oxidase.

[0016] In some embodiments of the present invention, the functional chaperone protein is a protein containing an iron porphyrin unit and other electron transfer properties. The protein can be used as a redox carrier to achieve electron transfer, and the redox potential of the functional chaperone protein is relatively low.

[0017] In some embodiments of the present invention, the functional chaperone protein includes at least one of cytochrome b562, cytochrome c, and cytochrome P450.

[0018] Cytochrome b562 is a small protein with a molecular weight of about 12.7KDa. The protein adopts a four-helix bundle folding and is coordinated with the heme iron molecule through ligands provided by Met7 and His102 located near the end of the polypeptide. It is considered to be a simple electron carrier that can shuttle electrons between the redox centers of other protein molecules. By fusing cytochrome b562 to NOx oxidase and using it as a redox electron carrier, efficient electron transport is established between the enzyme and the electrode, and direct electron transfer at low potential is achieved ( Figure 1 ).

[0019] In some embodiments of the present invention, the NOx oxidase includes enzymes that can undergo an oxidation reaction under the substrate NAD(P)H and oxygen, and a water-producing NAD(P)H oxidase whose byproduct is water.

[0020] In some embodiments of the present invention, the NOx oxidase has a FAD or FMN cofactor unit, including at least one of LmNOx derived from Leuconostoc mesenteroides, SmNOx derived from Streptococcus mutans, LbNOx derived from Lactobacillus belevyensis, and LsNOx derived from Lactobacillus sanfrancisco.

[0021] In some embodiments of the present invention, the functional chaperone protein is located at the N-terminus or C-terminus of the NOx oxidase.

[0022] In some embodiments of the present invention, the functional chaperone protein is directly linked to the NOx oxidase or is linked via a linker element.

[0023] In some embodiments of the present invention, the linking element connection is composed of 1 to 20 flexible amino acids, and the flexible amino acids are selected from at least one of Gly, Ser, Glu, Ala, Thr, and Lys.

[0024] In some embodiments of the present invention, the linker is a flexible linker, and the amino acid sequence of the linker includes but is not limited to GSAS, (GGCAGCGCCAGC) n 、(GGCGGCGGCAGC) n 、(GGCGGCGGCGGCAGC) n 、YAPVDV、(GGGS) n 、(GGSG) n 、(GGGGS) n , (G) n , where 1≤n≤5, and n is an integer.

[0025] In some embodiments of the present invention, the rigid linker comprises (EAAAK) n or (XP) n , where X can be any amino acid (Ala, Lys, Glu).

[0026] In some embodiments of the present invention, the cleavable linker comprises a 2A peptide, such as at least one of T2A, E2A and F2A.

[0027] In some embodiments of the present invention, the amino acid sequence of the functional chaperone protein is shown in SEQ ID NO:1.

[0028] In some embodiments of the present invention, the amino acid sequence of the NOx oxidase is shown in SEQ ID NO:3.

[0029] In some embodiments of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO:4.

[0030] The second aspect of the present invention provides a biological material related to the fusion protein of the first aspect of the present invention, wherein the biological material is any one of a1) to a12):

[0031] a1) a nucleic acid molecule encoding the fusion protein of the first aspect of the present invention;

[0032] a2) an expression cassette containing the nucleic acid molecule described in a1);

[0033] a3) a recombinant vector containing the nucleic acid molecule described in a1);

[0034] a4) a recombinant vector containing the expression cassette described in a2);

[0035] a5) a recombinant microorganism containing the nucleic acid molecule described in a1);

[0036] a6) a recombinant microorganism containing the expression cassette described in a2);

[0037] a7) a recombinant microorganism containing the recombinant vector described in a3);

[0038] a8) a recombinant microorganism containing the recombinant vector described in a4);

[0039] a9) a transgenic animal cell line containing the nucleic acid molecule described in a1);

[0040] a10) a transgenic animal cell line containing the expression cassette described in a2);

[0041] a11) a transgenic animal cell line containing the recombinant vector described in a3);

[0042] a12) A transgenic animal cell line containing the recombinant vector described in a4).

[0043] In some embodiments of the present invention, the expression cassette comprises a 5' transcription control region, an open reading frame encoding the fusion protein of the first aspect of the present invention, a translation control signal, a 3' untranslated region (3'UTR) and a transcription termination signal.

[0044] In some embodiments of the present invention, the 5' transcription control region comprises a promoter (a universal promoter, such as a viral promoter (SV40 promoter) or a mammalian "housekeeping" promoter, may be used), a transcription start site, an enhancer and / or a silencer element.

[0045] In some embodiments of the invention, the 3'UTR may encode an AU-rich element, which is a common regulator of mRNA stability through the 3'-5' exosome pathway and is usually located in the 3'UTR. The AU-rich element may contain one or more repeats of the sequence AUUUA. It may also contain one or more so-called US2B elements having the sequence AUAUAU.

[0046] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0047] In some embodiments of the present invention, the vector is independently selected from a non-pathogenic viral vector and a viral vector.

[0048] In some embodiments of the present invention, the viral vector comprises at least one of a lentiviral vector, an adenoviral vector, a baculoviral vector, a retroviral vector, a poxvirus vector, a Sendai virus vector, and a herpes simplex virus vector.

[0049] In some embodiments of the present invention, the non-viral vector includes at least one of a plasmid vector, a cationic polymer vector, chitosan, polyethyleneimine, a nanoparticle vector, and a liposome.

[0050] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a phage, a cosmid, a F cosmid, or an artificial chromosome.

[0051] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0052] In some embodiments of the present invention, the recombinant expression vector uses pET-28a(+) as the original expression vector.

[0053] The third aspect of the present invention provides use of the fusion protein of the first aspect of the present invention and / or the biomaterial of the second aspect of the present invention in the preparation of an enzyme electrode.

[0054] The fourth aspect of the present invention provides an enzyme electrode, comprising the fusion protein of the first aspect of the present invention and / or the biomaterial of the second aspect of the present invention.

[0055] In some embodiments of the present invention, the enzyme electrode further comprises a binder / cross-linking agent and a substrate material.

[0056] First, the functional chaperone protein, hydrophobic flexible linker and NOx oxidase are fused and expressed, and then the concentration of the fusion protein is increased by a binder / cross-linker and fixed to the electrode. As an electron transfer mediator between the NOx enzyme and the electrode, the chaperone protein can assist in the long-range electron transfer between the enzyme and the electrode. In essence, this method first completes the continuous oxidation regeneration of the cofactor FADH2 to FAD by electrochemical means, and then realizes the controllable and continuous oxidation of the substrate NADH under the action of NOx, thereby providing preliminary technical support for the synthesis and preparation of value-added chemicals through multi-enzyme cascade catalytic reactions. This method only requires the construction of a functional fusion protein gene to achieve mass production in Escherichia coli. The preparation of the enzyme electrode is completed through cross-linking and electrostatic adsorption steps, which is simple to operate and easy to implement.

[0057] In some embodiments of the present invention, the base electrode is not limited to a gold electrode or a glassy carbon electrode, and can be any electrode that can load an electrode solution, such as a carbon cloth electrode, a carbon paper electrode, a foamed nickel electrode, or a foamed copper electrode.

[0058] In some embodiments of the present invention, the cross-linking agent is a charged compound with good biocompatibility and cross-linking properties, which may be positively charged or negatively charged, including but not limited to chitosan and its derivatives, chitin and its derivatives, naphthol, dextran and its derivatives, glutaraldehyde, polyethyleneimine and / or polydimethyl ammonium chloride.

[0059] The fusion protein-based enzyme electrode preparation method realizes direct electron transfer at low potential, including utilizing the advantages of the partner protein in the fusion protein as an electron transfer mediator and its low redox potential to establish efficient electron transfer between the catalytic active center FAD / FADH2 and the electrode.

[0060] The fifth aspect of the present invention provides a method for preparing the enzyme electrode of the fourth aspect of the present invention, comprising the following steps:

[0061] A complex solution containing a cross-linking agent and the fusion protein is placed on the surface of a substrate material to form an enzyme electrode.

[0062] In some embodiments of the present invention, before the composite solution is placed on the surface of the substrate material, the substrate material is specifically modified using flexible bifunctional molecules to introduce positively or negatively charged groups on its surface.

[0063] In some embodiments of the present invention, the positively or negatively charged groups include, but are not limited to, hydroxyl groups, carboxyl groups and / or amino groups.

[0064] In some embodiments of the present invention, the substrate electrode modification method includes but is not limited to utilizing the coordination effect of a gold electrode and a thiol group (such as 11-mercaptoundecanoic acid (MUA) / 11-mercapto-1-undecanol (MU)) to self-assemble to form a monolayer (SAMs) to introduce negatively charged groups on the electrode surface. The specific implementation process is as follows: a gold electrode is placed in an ethanol solution containing a thiol bifunctional molecule and placed at room temperature for 20 to 30 hours to perform anion modification on the surface of the gold electrode.

[0065] In some embodiments of the present invention, the final concentration of the cross-linking agent in the composite solution is 0.1% to 1%.

[0066] In some embodiments of the present invention, the final concentration of the fusion protein in the composite solution is 10-15 mg / mL.

[0067] In some embodiments of the present invention, the mixing conditions are: first, high-speed mixing at room temperature for about 15 to 30 seconds, and then incubation at 4° C. for 20 minutes to form a uniform mixed solution.

[0068] In some embodiments of the present invention, the composite solution is coated on the surface of a substrate material and dried and solidified to form an enzyme electrode.

[0069] A sixth aspect of the present invention provides a bioelectrochemical reactor, wherein the bioelectrochemical reactor adopts a three-electrode system, including a working electrode, a reference electrode and a counter electrode, wherein the working electrode is the enzyme electrode of the fifth aspect of the present invention.

[0070] In some embodiments of the present invention, the reference electrode may be a saturated calomel electrode (SCE) or a silver-silver chloride (Ag / AgCl) electrode, and the counter electrode may be a platinum sheet / wire electrode or a carbon rod electrode (including but not limited to a graphite electrode).

[0071] In some embodiments of the present invention, the enzyme electrochemical reactor further comprises an electrolytic cell and an electrolyte solution.

[0072] In some embodiments of the present invention, the electrolyte solution includes a substrate.

[0073] In some embodiments of the invention, the substrate comprises NAD(P)H.

[0074] In some embodiments of the present invention, the electrolytic cell in the electrochemical reactor uses a proton exchange membrane to separate the anode chamber and the cathode chamber.

[0075] In some embodiments of the present invention, the electrolyte base solution includes but is not limited to PBS buffer.

[0076] The seventh aspect of the present invention provides a method for preparing oxidized NAD+ or NAD(P)+, comprising the step of using the bioelectrochemical reactor of the sixth aspect of the present invention to carry out a reaction to prepare oxidized NAD+ or NAD(P)+.

[0077] Among them, if the substrate of the fusion protein (NOx oxidase) is NADPH, NADP+ is prepared by reacting through the enzyme electrochemical reactor of the sixth aspect of the present invention; if the substrate of NOx oxidase is NADH, NAD+ is prepared by reacting through the enzyme electrochemical reactor of the sixth aspect of the present invention; some water-producing NOx (such as LsNOx) have substrates of NAD and NADPH, and NAD+ and NADP+ are prepared by reacting through the electrochemical reactor described in the sixth aspect of the present invention.

[0078] The beneficial effects of the present invention are:

[0079] Since the catalytic active center of the oxidoreductase is deeply buried inside its structure, it is difficult to form an effective electron exchange between the active center and the electrode. The present invention provides a fusion protein composed of NOx oxidase and a functional chaperone protein, which can serve as an intermediate carrier for electron transfer and has a low electrochemical potential, and can assist in realizing electron transfer between the enzyme and the electrode. The invention provides important technical support for achieving efficient and controllable coenzyme regeneration, and at the same time provides a preliminary foundation for the application of multi-enzyme cascades in the preparation and synthesis of high value-added chemicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 Design of fusion protein (A), iron-porphyrin structure of cytochrome b562 (B), and corresponding bioelectrocatalytic oxidation system (C).

[0081] Figure 2 The biochemical characteristics of wild-type LmNOx and b562-linker1-LmNOx are analyzed; A is a digital photo; B is the SDS-PAGE analysis result of wild-type LmNOx and b562-linker1-LmNOx, in which 1 is wild-type LmNOx and 2 is b562-linker1-LmNOx; C is the mass spectrometry analysis result of wild-type LmNOx; D is the mass spectrometry analysis result of b562-linker1-LmNOx; E is the optimum pH value determination result of wild-type LmNOx; F is the anti-interference ability test result of wild-type LmNOx; G is the kinetic parameters of wild-type LmNOx determined by isothermal titration calorimetry (ITC). number; H is the optimum pH value determination result of b562-linker1-LmNOx; I is the anti-interference ability test result of b562-linker1-LmNOx; J is the kinetic parameters of b562-linker1-LmNOx determined by isothermal titration calorimetry (ITC); K is the circular dichroism (CD) spectra of LmNOx wild type and b562-linker1-LmNOx; L is the differential scanning fluorescence (DSF) and static light scattering (SLS266) analysis results of LmNOx wild type; N is the differential scanning fluorescence (DSF) and static light scattering (SLS266) analysis results of b562-linker1-LmNOx.

[0082] Figure 3 Flowchart for enzyme electrode preparation.

[0083] Figure 4Characterization of enzyme immobilization; wherein, A and B are scanning electron microscopy (SEM) images of wild-type LmNOx; C and D are SEM images of b562-linker1-LmNOx; E, I and M are confocal laser scanning microscopy (CLSM) images of wild-type LmNOx; F and J are atomic force microscopy (AFM) images of wild-type LmNOx bound to CTS before and after immobilization; G, K and O are CLSM images of b562-linker1-LmNOx; H and L are AFM images of b562-linker1-LmNOx bound to CTS before and after immobilization; N is DLS analysis of wild-type LmNOx / CTS complex; P is DLS analysis of b562-linker1-LmNOx / CTS complex.

[0084] Figure 5 Characterization of the DET properties of the fusion protein b562-linker1-LmNOx; wherein A is a comparison of the CV curves of the wild type and the fusion protein before and after the addition of NADH in a pH 5.0 electrolyte solution; B is a comparison of the CV curves of the fusion protein before and after the addition of NADH in a pH 7.4 electrolyte solution. DETAILED DESCRIPTION

[0085] The present invention is further described in detail below through specific examples.

[0086] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0087] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0088] The technical solution of the present invention:

[0089] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0090] Example 1 Preparation of fusion protein based on cytochrome b562 chaperone protein

[0091] Fusion protein gene cloning: Using the synthetic pET28a(+)-Cytochrome b562 plasmid as the template gene, primers at both ends of the cytochrome b562 gene (Uniprot ID is P0ABE7) were designed, and a flexible linker was designed and introduced. 2×Phanta Max Master Mix enzyme was used for PCR amplification to obtain the Cytochrome b562 gene containing linkers of different lengths. At the same time, the recombinant pColdⅠDNA-LmNOx (LmNOx's Uniprot ID is Q03UI6) was used as a template plasmid to obtain a linearized vector fragment by PCR. The ClonExpress MultiS one-step cloning kit was then used to splice the target gene and the vector fragment at 37°C for 30 min. The spliced ​​gene fragment was transformed into Escherichia coli DH5α competent cells, and the cloned strain was picked and sequenced to obtain the target gene.

[0092] Among them, the amino acid sequence of Cytochrome b562 is MADLEDNMETLNDNLKVIEKADNAAQVKDALTKMRAAALDAQKATPPKLEDKSPD SPEMKDFRHGFDILVGQIDDALKLANEGKVKEAQAAAEQLKTTRNAYHQKYR (SEQ ID NO: 1);

[0093] The amino acid sequence of the linker is GGSGGSGGSGGSGGS (SEQ ID NO: 2);

[0094] The amino acid sequence of LmNOx is MKIGIIGATHAGVYAAKQIKSEHPEAEVTVFEKNTTVSFLSCGIALWIGDHVSDPEKMFYETPESMRNQGITMLMAHQVNQADLKSKTLTVTNLDTEEVTTQQFDKIVITTGSKPVIPNIPGIDSNRVYFCKSWDDATRIKSVAKDLKRVAVIGAGYIGAELAEQLSVTGKQVTLIDGADRVLAKNFGPAFTERIQKEYEKNNVDVVLNQFVSKFENTPNG IKITTPNGSYEADIAILGIGFLPNTDLFSGQLETLPNGAIIVNDYMQTSEDNVYAAGDASTVFFNPTQAIDYIPLATNAIRQGILVGKNIVEPTTRYLGTQATSAVELYEQAMVATG LNSATAKEKGLDVIETVIESDYRPDFMLSTTSVLSSLTWEKDTGRIVGGSFMSQHDVSQSANLVSIAIQKQMTIDELAMSDFFFQPNFSQPINYIPEVALAAVAEKQALTLAY(SEQ ID NO:3).

[0095] Preparation of E. coli expression strain: The plasmid identified by sequencing was heat-treated at 42°C and transformed into BL21 (DE3) competent cells, revived by shaking culture at 37°C and spread on LB plates containing kanamycin for screening. A single colony was picked as the E. coli expression strain, expanded and cultured in liquid LB medium (containing 50 μg / mL ampicillin) at 37°C, 200 rpm to extract the plasmid, and stored in glycerol stock at –80°C for later use.

[0096] Protein expression: The frozen glycerol bacteria were inoculated into LB liquid medium containing kanamycin and cultured overnight at 37°C and 200 rpm to activate the strain. Then, 2% of the activated bacterial solution was inoculated into fresh LB liquid medium containing kanamycin and cultured until the bacterial solution OD 600 When the pH reaches 0.4-0.6, the bacteria enter the logarithmic growth phase. Then, 0.2 mM isopropyl-β-D-thiogalactoside inducer is added to induce high-speed protein expression at 15°C and 180 rpm for 24 hours.

[0097] Preparation of fusion protein: The strain after expression was centrifuged at 4°C, 8000rpm for 15min to collect the bacteria, and the supernatant LB medium was discarded. After adding an appropriate amount of binding buffer (20mM Tris-HCl, 150mM sodium chloride, 20mM imidazole) to suspend the bacteria, the cells were broken by a high-pressure homogenizer, and then centrifuged at 4°C, 20000rpm for 30min to remove insoluble proteins and cell debris, etc., to obtain a soluble crude protein supernatant. The HisTrap excel column of the protein liquid phase purification system AKTA pure was used to separate and purify the target protein, and the nickel filler in the column was used to specifically bind to the histidine tag in the fusion protein to remove impurities. Subsequently, a linear gradient elution was performed using an elution buffer (20mM Tris-HCl, 150mM sodium chloride, 500mM imidazole). A large amount of imidazole will competitively bind to the nickel filler to gradually elute the histidine-tagged fusion protein from the column. The obtained protein was desalted by PD-10 column to remove small molecules such as imidazole and salts to obtain the purified target protein. Finally, the purified protein was identified by SDS-PAGE electrophoresis, aliquoted and quickly frozen in liquid nitrogen, and stored at -80°C for future use.

[0098] Figure 2 Figure A is a digital photo of the two proteins, in which the fusion protein visually appears obvious red, which is the characteristic color of Heme, the functional unit of cytochrome b562, thus visually confirming the successful expression of the fusion. In addition, Figure 2 The SDS-PAGE and mass spectrometry results of B to D in the figure further confirmed the above conclusion. The results showed that compared with the wild-type LmNOx, the fusion protein (denoted as b562-linker1-LmNOx) had a larger molecular weight of about 64KDa, which was almost consistent with the predicted size. In addition, SDS-PAGE showed that its purity was >95%.

[0099] The amino acid sequence of b562-linker1-LmNOx is (SEQ ID NO: 4).

[0100] like Figure 2 Figures E to N show a detailed comparison of the biochemical properties of b562-linker1-LmNOx and the wild type. Figure 2 Figures E and H in Figure 2 show that the catalytic activity of NOx before and after fusion has hardly changed significantly (about 100 U / mg), and the optimal pH and activity change trends are consistent. Figure 2 We tested the anti-interference ability of 2 in F and 2 in I before and after fusion, and the results showed that the fusion proteins also had good stability. Then, we used isothermal titration calorimetry to measure the kinetic parameters of the two to quantitatively explain the differences before and after fusion. Figure 2 As shown in G and J, the k of wild-type and fusion proteins cat / K MThe above results clearly show that the fusion of Cytochrome b562 to NOx does not significantly affect its original activity. In addition, in order to further understand the above results, we analyzed the circular dichroism (CD) spectrum, differential scanning fluorimetry (DSF) and static light scattering (SLS266) of the two to analyze them structurally. Figure 2 The K and CD results showed that the secondary structure of the fusion protein did not change significantly compared with the wild type. Figure 2 Figures 2L and 2N show that DSF and SLS analysis compared the differences in their Tm and Tagg in detail.

[0101] Example 2 Preparation of enzyme electrode

[0102] Preparation of enzyme electrode Figure 3 As shown, the specific preparation process is as follows: First, the gold electrode is polished in sequence using metallographic sandpaper and Al2O3 polishing powder of different particle sizes. After each polishing, it is thoroughly cleaned with deionized water before the next polishing. After polishing, the electrode is ultrasonically cleaned in acetone, ethanol and deionized water for 10 minutes in turn to remove surface oxides and impurities as much as possible. Subsequently, the cleaned gold electrode is placed in a 0.5M H2SO4 solution and electrochemically activated at room temperature for standby use. Then, the cleaned gold electrode is placed in a 100mM 11-mercaptoundecanoic acid / 11-mercapto-1-undecyl alcohol (MUA / MU) solution containing ethanol at a ratio of 1:3 and placed at room temperature for 24 hours to modify the surface of the gold electrode with anions. Next, 0.5% cellulose polysaccharide chitosan (CTS) solution and a certain concentration of b562-linker1-LmNOx solution are mixed quickly in equal volumes and left to stand at 4°C for about 20 minutes to ensure that the two are fully mixed. Finally, 5 μL of the mixed solution was dropped onto the surface of the modified gold electrode and dried at 4° C. to obtain the enzyme electrode (b562-linker1-LmNOx).

[0103] The enzyme electrode of wild-type LmNOx was prepared according to the above method, except that b562-linker1-LmNOx was replaced by wild-type LmNOx.

[0104] Example 3

[0105] In this example, the enzyme electrode prepared in Example 2 is characterized as follows:

[0106] The electrode solutions of wild-type and fusion proteins were compared. Figure 4As shown. First, the change in particle size after enzyme immobilization was shown by dynamic light scattering DLS. At the same time, the prepared electrode solution was characterized by morphology. The confocal laser scanning microscope (CLSM) sample preparation method was to take 10μL of sample and directly drop it on a glass slide, and fix the sample with a cover glass to place it through a scanning electron microscope, and a 60x oil-immersion objective was used. The specific scanning parameters were power 75mW, intensity 30%, exposure time 510ms, and imaging under white light and blue light respectively. The blue light was excited by 445nm excitation light, and the image was collected by point scanning. In addition, the scanning electron microscope (SEM) sample preparation was to first dry the electrode composite solution naturally and collect images at an accelerating voltage of 3kV. The sample preparation for the atomic force microscope (AFM) was to directly dilute the prepared electrode solution, carefully drip it on the freshly torn mica sheet and quickly cover the mica sheet.

[0107] Figure 4 Figures A to D are SEM images of the wild type and fusion proteins. Both of them form smaller block structures under the action of chitosan, and are intertwined and connected to each other, eventually forming a porous quasi-three-dimensional network structure. This structure is expected to significantly enhance the catalytic effect in the subsequent catalytic process. Figure 4 4 in E, 4 in I and 4 in M, and Figure 4 Figures 4G, 4K, and 4O show the CLSM images of the wild-type and fusion proteins under the action of chitosan. The results clearly show that the two are evenly dispersed in the chitosan solution and exist stably. Subsequently, to confirm the cross-linking effect of chitosan in the immobilization process, we compared the AFM and DLS changes of the two before and after the addition of chitosan in detail. For the wild-type, Figure 4 Figure J clearly shows that the size of the protein in the field of view increased significantly after the addition of chitosan. Figure 4 The DLS results of N in the samples further confirmed this conclusion. Figure 4 As shown in Figures 4H, 4L, and 4P, the AFM and DLS of the fusion protein also showed similar phenomena, but the morphology was slightly different. The above results indicate that b562-linker1-LmNOx has different structures and surface properties from the wild type during immobilization, which may affect its electrocatalytic performance.

[0108] Example 4

[0109] In this example, the electrochemical performance of the enzyme electrode prepared in Example 2 was tested as follows:

[0110] By using a saturated calomel electrode (SCE) as a reference electrode, a platinum electrode as a counter electrode, and the enzyme-loaded gold electrode in Example 2 as a working electrode, we systematically studied the electrochemical properties of the fusion protein, focusing on verifying its direct electron transfer (DET) process. The experiment used cyclic voltammetry (CV) to analyze the effective electron transfer between the b562 domain of the fusion protein and the electrode, especially under different pH conditions (pH 5.0 and 7.4). The pH dependence of DET was explored in depth. Under pH 5.0, the experimental results are as follows Figure 5 As shown in Figure A, there is no significant difference in the CV curves of the wild type and fusion protein when NADH is not added, and no obvious redox peaks are observed. However, after the addition of 2 mM NADH, the fusion protein produces a significant electrocatalytic current at about 0.6 V (vs. RHE), which is more prominent than the wild type. This phenomenon strongly suggests an effective electron transfer process between FAD / FADH2 and the electrode mediated by the b562 domain. In contrast, under pH 7.4 ( Figure 5 (B) Even after the addition of NADH, the CV curve of the fusion protein remained almost unchanged, further confirming that the increase in electrocatalytic current was caused by the mediation of cytochrome b562 rather than other factors.

[0111] The reason for the difference in pH can be attributed to the effect of pH on intramolecular electron transfer (IET), that is, the electron transfer channel between Heme (Fe2+ / Fe3+) and FAD / FADH2. At pH 5.0, the amino acid residues around FAD / FADH2 are protonated, making its internal region positively charged; while at pH 7.4, the amino acid residues around FAD / FADH2 may be partially deprotonated, which will make its internal region negatively charged. In contrast, existing literature has shown that the charge distribution around Heme (Fe2+ / Fe3+) exhibits negative charge characteristics at both pH 5.0 and pH 7.4. Therefore, at pH 7.4, since FAD / FADH2 and Heme have the same charge, the electrostatic repulsion between the two leads to low efficiency of intramolecular electron transfer, and thus no significant oxidation current is generated.

[0112] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A fusion protein comprising a functional chaperone protein and a NOx oxidase.

2. The fusion protein according to claim 1, characterized in that The functional chaperone protein is a protein containing an iron porphyrin functional unit and other proteins with electron transfer properties; and / or, the NOx oxidase includes enzymes that can undergo oxidation reactions under the substrate NAD(P)H and oxygen.

3. The fusion protein according to claim 2, characterized in that The functional chaperone protein includes at least one of any variant forms of heme b, heme b562, cytochrome c, and cytochrome P450 iron-containing porphyrin structural units; and / or the NOx oxidase has a FAD or FMN cofactor unit, including LmNOx derived from Leuconostoc mesenteroides, SmNOx derived from Streptococcus mutans, LbNOx derived from Lactobacillus belivi, LsNOx derived from Lactobacillus sanfrancisco, and at least one of other homologous sources.

4. The fusion protein according to any one of claims 1 to 3, characterized in that The functional chaperone protein is located at the N-terminus or C-terminus of the NOx oxidase; and / or, and / or, the functional chaperone protein is directly connected to the NOx oxidase or connected via a linking element; Preferably, the linking elements include flexible linkers, rigid linkers and shearable linkers; Preferably, the amino acid sequence of the functional chaperone protein is as shown in SEQ ID NO: 1; Preferably, the amino acid sequence of the NOx oxidase is as shown in SEQ ID NO:

3.

5. A biological material related to the fusion protein according to any one of claims 1 to 4, wherein the biological material is any one of a1) to a12): a1) a nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 4; a2) an expression cassette containing the nucleic acid molecule described in a1); a3) a recombinant vector containing the nucleic acid molecule described in a1); a4) a recombinant vector containing the expression cassette described in a2); a5) a recombinant microorganism containing the nucleic acid molecule described in a1); a6) a recombinant microorganism containing the expression cassette described in a2); a7) a recombinant microorganism containing the recombinant vector described in a3); a8) a recombinant microorganism containing the recombinant vector described in a4); a9) a transgenic animal cell line containing the nucleic acid molecule described in a1); a10) a transgenic animal cell line containing the expression cassette described in a2); a11) a transgenic animal cell line containing the recombinant vector described in a3); a12) A transgenic animal cell line containing the recombinant vector described in a4).

6. Use of the fusion protein according to any one of claims 1 to 4 and / or the biomaterial according to claim 5 in the preparation of an enzyme electrode.

7. An enzyme electrode comprising the fusion protein according to any one of claims 1 to 4 and / or the biomaterial according to claim 5; Preferably, the enzyme electrode further comprises a cross-linking agent and a substrate material; Preferably, the substrate material comprises a gold electrode, a glassy carbon electrode, a carbon cloth electrode, a carbon paper electrode, a foamed nickel electrode or a foamed copper electrode; Preferably, the cross-linking agent comprises chitosan, chitin, naphthol, dextran, glutaraldehyde, polyethyleneimine and / or polydiallyl dimethyl ammonium chloride.

8. The method for preparing the enzyme electrode according to claim 7, comprising the following steps: placing a composite solution containing a cross-linking agent and the fusion protein on the surface of a substrate material to form an enzyme electrode; Preferably, the substrate material is modified before the composite solution is placed on the surface of the substrate material to introduce positively or negatively charged groups; Preferably, the positively or negatively charged groups include carboxyl groups and / or amino groups.

9. A bioelectrochemical reactor, wherein the bioelectrochemical reactor adopts a three-electrode system, including a working electrode, a reference electrode and a counter electrode, wherein: The working electrode is the enzyme electrode according to claim 7.

10. A method for preparing oxidized NAD+ or NAD(P)+, comprising the step of using the bioelectrochemical reactor according to claim 9 to carry out a reaction to prepare oxidized NAD+ or NAD(P)+.

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