An immobilized enzyme electrode and its preparation method and application

Abstract

The present invention belongs to the field of immobilized enzyme technology, and specifically relates to an immobilized enzyme electrode and its preparation method and application. The immobilized enzyme electrode provided by the present invention comprises a conductive carrier and a ferredoxin-NADP+ reductase and a reaction enzyme loaded on the conductive carrier; the conductive carrier comprises a conductive substrate and a porous graphene layer arranged on the surface of the conductive substrate, and the pore structure of the porous graphene layer is nanopores; the ferredoxin-NADP+ reductase and the reaction enzyme are loaded in the pore structure of the porous graphene layer. The immobilized enzyme electrode provided by the present invention can realize NADPH cyclic regeneration under the condition of an external power supply, ensure that the enzyme cascade reaction is continuous and stable; and the immobilized enzyme electrode can be used repeatedly, effectively reducing the cost of the enzyme cascade reaction, and is suitable for industrial expansion and application.

Description

Technical Field

[0001] The invention belongs to the technical field of immobilized enzymes, and in particular relates to an immobilized enzyme electrode and a preparation method and application thereof. Background Art

[0002] Enzyme cascade reactions are not only ubiquitous in nature, but also have important industrial applications. Generally, enzyme cascade reactions involve three basic components: multiple enzymes, substrates, and products. The initial enzyme catalyzes the conversion of the substrate into a second substrate, which is then acted upon by the second enzyme, leading to the formation of a third substrate, and so on, until the final product is obtained. Enzyme cascade reactions require the participation of multiple enzymes, but since free enzymes are unstable and easily inactivated, they cannot be reused. Only immobilized enzymes are of practical value. The main methods for enzyme immobilization are adsorption. Adsorption refers to the adsorption of active enzymes on the surface of a carrier. Currently, commonly used enzyme immobilization carrier materials such as chitosan, bentonite, and silica gel have poor enzyme immobilization effects. The immobilized enzymes easily fall off the carrier, have poor stability and reproducibility, and lack conductivity.

[0003] On the other hand, enzyme-catalyzed redox, cleavage, isomerization, and transfer reactions generally require the participation of coenzyme factors. Generally, there are two ways to maintain coenzyme factor concentrations in industrial enzyme cascade reactions. One is to directly add coenzyme factors exogenously, but this will increase production costs; the other is to utilize glucose dehydrogenase or formate dehydrogenase to achieve coenzyme factor recycling. For example, reduced nicotinamide adenine dinucleotide phosphate (NADPH) is the most important coenzyme factor. Traditionally, the NADPH cycle utilizes glucose dehydrogenase (GDH), with glucose being continuously oxidized by GDH as a consumer. However, this process continuously produces gluconic acid as a byproduct, which not only reduces product purity but also causes the pH value of the reaction system to continuously decrease, affecting the activity of other enzymes, making continuous industrial production impossible.

[0004] In summary, in order to reduce the cost of industrial enzyme cascade reactions and achieve scale-up production, it is urgent to develop an immobilized enzyme reaction system that can realize the circulation of coenzyme factors under a mild environment without introducing too many by-products. Summary of the Invention

[0005] The purpose of the present invention is to provide an immobilized enzyme electrode and its preparation method and application. The immobilized enzyme electrode provided by the present invention can directly use electrochemistry to reduce nicotinamide adenine dinucleotide phosphate (NADP+), achieving stable cyclic regeneration of the coenzyme NADPH without generating by-products, and has high stability, which can effectively reduce the cost of enzyme cascade reactions and is suitable for industrial expansion and application.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides an immobilized enzyme electrode, comprising a conductive carrier and a ferredoxin-NADP+ reductase and a reaction enzyme loaded on the conductive carrier; the conductive carrier comprises a conductive substrate and a porous graphene layer arranged on the surface of the conductive substrate, the pore structure of the porous graphene layer being nanopores; the ferredoxin-NADP+ reductase and the reaction enzyme are loaded in the pore structure of the porous graphene layer; the reaction enzyme is one or more of an oxidoreductase, a lyase, an isomerase and a transferase.

[0008] Preferably, the reaction enzyme is an oxidoreductase.

[0009] Preferably, the mass ratio of the ferredoxin-NADP+ reductase to the reaction enzyme is 1:(0.1-10).

[0010] Preferably, the reaction enzyme is glutamate dehydrogenase or alcohol dehydrogenase.

[0011] Preferably, the method for preparing the conductive carrier comprises the following steps:

[0012] mixing graphene, glacial acetic acid, a first solvent, and a co-solvent to obtain a graphene dispersion;

[0013] mixing a binder and a second solvent to obtain a binder solution;

[0014] The graphene dispersion and the binder solution are mixed and concentrated to obtain a graphene slurry;

[0015] coating the graphene slurry on a surface of a conductive substrate, and heat-treating the coated conductive substrate to obtain a conductive carrier;

[0016] The binder is selected from one or more of ethyl cellulose, hydroxyethyl cellulose, methyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polyurethane, polyacrylic acid, polymethyl methacrylate, polyethyl methacrylate, polyvinyl alcohol and derivatives of the above compounds; the cosolvent is terpineol;

[0017] The heat treatment includes a first heat treatment and a second heat treatment performed in sequence, wherein the temperature of the first heat treatment is 50-100°C, and the temperature of the second heat treatment is 300-500°C.

[0018] The present invention provides a method for preparing the immobilized enzyme electrode described in the above technical solution, comprising the following steps:

[0019] The ferredoxin-NADP+ reductase solution is coated on the surface of the conductive support and allowed to stand for a first time to obtain a first loaded electrode;

[0020] The reaction enzyme solution is coated on the surface of the first load electrode, and the immobilized enzyme electrode is obtained after a second static state; the temperature of the first static state and the second static state are independently ≤5°C.

[0021] Preferably, the insulation time of the first standing and the second standing are independently 20 to 30 minutes.

[0022] Preferably, the mass concentration of the ferredoxin-NADP+ reductase solution is 10 to 20 mg / mL.

[0023] Preferably, the mass concentration of the reaction enzyme solution is 5 to 15 mg / mL.

[0024] The present invention provides the use of the immobilized enzyme electrode described in the above technical solution or the immobilized enzyme electrode prepared by the preparation method described in the above technical solution in electrocatalytic enzyme cascade reaction.

[0025] The present invention provides an immobilized enzyme electrode, comprising a conductive carrier and a ferredoxin-NADP+ reductase and a reaction enzyme loaded on the conductive carrier; the conductive carrier comprises a conductive substrate and a porous graphene layer arranged on the surface of the conductive substrate, the pore structure of the porous graphene layer being nanopores; the ferredoxin-NADP+ reductase and the reaction enzyme are loaded in the pore structure of the porous graphene layer; the reaction enzyme is one or more of an oxidoreductase, a lyase, an isomerase and a transferase. The present invention adopts the nanoscale pore structure of porous graphene layer to fix ferredoxin-NADP+ reductase and reaction enzyme, and each nanopore channel that fixes enzyme is used as a nano-microreactor, under the effect of external power supply, can utilize the electron provided by external power supply to directly realize NADP+ to generate NADPH, thereby realizing NADPH cycle regeneration, and will not introduce by-products into the reaction system, ensuring the stable progress of the reaction; At the same time, the present invention disperses and fixes ferredoxin-NADP+ reductase and reaction enzyme in nanopore channel, not only improves the fixation stability of enzyme molecules, enzyme molecules are not easy to aggregate, and under the protection of nanopore channel, enzyme molecules can withstand more severe reaction conditions, such as more extreme temperature (50 DEG C), pH value and pressure, thereby improving the application range of fixed enzyme electrode. In summary, the fixed enzyme electrode provided by the present invention can realize NADPH cycle regeneration under the condition of applied voltage, ensure that enzyme cascade reaction is continuous and stably carried out, and the fixed enzyme electrode can be used repeatedly, effectively reducing the cost of enzyme cascade reaction, and is suitable for industrial expansion application.

[0026] Furthermore, in the present invention, the preparation method of the conductive carrier includes the following steps: mixing graphene, glacial acetic acid, a first solvent and a co-solvent to obtain a graphene dispersion; mixing a binder and a second solvent to obtain a binder solution; mixing the graphene dispersion and the binder solution and concentrating them to obtain a graphene slurry; coating the graphene slurry on the surface of a conductive substrate, and heat-treating the coated conductive substrate to obtain a conductive carrier; the binder is selected from one or more of ethyl cellulose, hydroxyethyl cellulose, methyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polyurethane, polyacrylic acid, polymethyl methacrylate, polyethyl methacrylate, polyvinyl alcohol and derivatives of the above compounds; the co-solvent is terpineol; the heat treatment includes performing a first heat treatment and a second heat treatment in sequence, the temperature of the first heat treatment is 50-100°C, and the temperature of the second heat treatment is 300-500°C. When the present invention prepares a conductive carrier, a specific binder, a cosolvent and glacial acetic acid are added to the graphene slurry, wherein the binder can make graphene and the electrode substrate firmly compounded, and the glacial acetic acid can improve the wetting and spreading of the graphene coating on the surface of the electrode substrate, improve the overall quality and consistency of the coating, and the cosolvent helps to form a uniform graphene coating, and the binder can play a supporting role to the nanopores of graphene, avoiding damage to the graphene nanopores; In addition, the present invention performs a step-by-step heat treatment on the conductive substrate coated with the graphene slurry, which can fully eliminate defects such as bubbles in the graphene coating and improve the bonding force between the graphene coating and the electrode substrate. Therefore, the preparation method provided by the present invention can firmly compound graphene with the electrode substrate, form a firm graphene coating on the substrate surface, and form a nanopore structure. The conductive carrier prepared by the present invention has a high specific surface area and a superior nanopore structure, which is more conducive to the stable load of enzyme molecules and NADPH cyclic regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a physical picture of the conductive carrier prepared in Example 1 of the present invention;

[0028] Figure 2 This is an electron microscope image of the conductive carrier prepared in Example 1 of the present invention;

[0029] Figure 3 This is a curve showing the change of the current over time in the enzyme-catalyzed reaction in Example 1 of the present invention;

[0030] Figure 4 This is the hydrogen nuclear magnetic resonance spectrum of the enzyme-catalyzed reaction solution of Example 1 of the present invention before the reaction;

[0031] Figure 5 This is the hydrogen nuclear magnetic resonance spectrum of the enzyme-catalyzed reaction solution of Example 1 of the present invention after 18 hours of reaction;

[0032] Figure 6This is a curve showing the change of the current over time in the enzyme-catalyzed reaction in Example 2 of the present invention;

[0033] Figure 7 This is the hydrogen nuclear magnetic resonance spectrum of the enzyme-catalyzed reaction solution of Example 2 of the present invention before the reaction;

[0034] Figure 8 This is the hydrogen nuclear magnetic resonance spectrum of the enzyme-catalyzed reaction solution of Example 2 of the present invention after 16 hours of reaction;

[0035] Figure 9 This is a curve showing the change of the current over time in the enzyme-catalyzed reaction in Example 3 of the present invention;

[0036] Figure 10 This is a schematic diagram of the reaction principle of the immobilized enzyme electrode of the present invention. DETAILED DESCRIPTION

[0037] The present invention provides an immobilized enzyme electrode, comprising a conductive carrier and a ferredoxin-NADP+ reductase and a reaction enzyme loaded on the conductive carrier; the conductive carrier comprises a conductive substrate and a porous graphene layer arranged on the surface of the conductive substrate, the pore structure of the porous graphene layer being nanopores; the ferredoxin-NADP+ reductase and the reaction enzyme are loaded in the pore structure of the porous graphene layer; the reaction enzyme is one or more of an oxidoreductase, a lyase, an isomerase and a transferase.

[0038] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0039] The enzyme-immobilized electrode provided by the present invention comprises a conductive carrier comprising a conductive substrate and a porous graphene layer arranged on the surface of the conductive substrate, wherein the pore structure of the porous graphene layer is nanopores.

[0040] In the present invention, the thickness of the porous graphene layer on the surface of the conductive support is preferably 10 to 50 μm, more preferably 15 to 45 μm. The pore size of the porous graphene layer is preferably 50 to 150 nm.

[0041] In the present invention, the method for preparing the conductive carrier preferably comprises the following steps:

[0042] mixing graphene, glacial acetic acid, a first solvent, and a co-solvent to obtain a graphene dispersion;

[0043] mixing a binder and a second solvent to obtain a binder solution;

[0044] The graphene dispersion and the binder solution are mixed and concentrated to obtain a graphene slurry;

[0045] coating the graphene slurry on a surface of a conductive substrate, and heat-treating the coated conductive substrate to obtain a conductive carrier;

[0046] The binder is selected from one or more of ethyl cellulose, hydroxyethyl cellulose, methyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polyurethane, polyacrylic acid, polymethyl methacrylate, polyethyl methacrylate, polyvinyl alcohol and derivatives of the above compounds; the cosolvent is terpineol;

[0047] The heat treatment includes a first heat treatment and a second heat treatment performed in sequence, wherein the temperature of the first heat treatment is 50-100°C, and the temperature of the second heat treatment is 300-500°C.

[0048] The present invention mixes graphene, glacial acetic acid, the first solvent and a cosolvent to obtain a graphene dispersion liquid. In the present invention, the graphene preferably comprises one or more of monolayer graphene, double-layer graphene, multilayer graphene, graphene oxide, hydrogenated graphene, fluorinated graphene, nitrogen-doped graphene, graphene film, graphene sheet, graphene quantum dots, graphene nanoribbons and three-dimensional graphene. In the present invention, the cosolvent is terpineol, which can be a mixture (industrially directly referred to as terpineol) of α-terpineol, β-terpineol, γ-terpineol, δ-terpineol or above configuration terpineol, preferably α-terpineol. In the present invention, the first solvent preferably includes one or more of water, acetone, methanol, ethanol, ethylene glycol, kerosene, diesel, tetrahydrofuran (THF), N-methylpyrrolidone (NMP), nitromethane and N,N-dimethylformamide (DMF); in an embodiment of the present invention, it is preferably a mixed solvent of water and acetone or a mixed solvent of water and N-methylpyrrolidone, and the volume ratio of water to acetone or the volume ratio of water to N-methylpyrrolidone in the mixed solvent is preferably 1:21.5; the water is preferably deionized water or ultrapure water. In the present invention, the usage ratio of the graphene to the first solvent is preferably (0.3-1) g:100 mL, more preferably (0.3-0.6) g:100 mL; the usage ratio of the co-solvent to the first solvent is preferably (1-3) g:100 mL, more preferably (2-2.5) g:100 mL; the volume ratio of the glacial acetic acid to the first solvent is preferably 0.1-0.5:100, more preferably 0.1-0.2:100.

[0049] In the present invention, the method for mixing the graphene, glacial acetic acid, the first solvent, and the co-solvent is preferably as follows: glacial acetic acid and the first solvent are sequentially added to the graphene for a first mixing, and the co-solvent is added to the resulting mixture for a second mixing to obtain a graphene dispersion. In the present invention, the first solvent is preferably added in portions. In an embodiment of the present invention, water is first added in portions, and then acetone or N-methylpyrrolidone is added in portions. The first and second mixing methods are preferably ultrasound, vibration, stirring, grinding, or vortexing. The time for the first and second mixing is based on sufficient dispersion of the components.

[0050] In the present invention, the cosolvent helps to form a uniform graphene coating; the glacial acetic acid can be adsorbed on the graphene surface, and the protons of the glacial acetic acid adsorbed on the graphene surface can change the Zeta potential to positive, which is beneficial to the suspension and dispersion of graphene in the solvent due to the effect of electrostatic repulsion.

[0051] The present invention mixes a binder and a second solvent to obtain a binder solution. In the present invention, the binder is selected from one or more of ethyl cellulose, hydroxyethyl cellulose, methyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polyurethane, polyacrylic acid, polymethyl methacrylate, polyethyl methacrylate, polyvinyl alcohol and derivatives of the above compounds, more preferably ethyl cellulose. In the present invention, the second solvent preferably includes one or more of water, acetone, methanol, ethanol, ethylene glycol, kerosene, diesel, tetrahydrofuran, N-methylpyrrolidone, nitromethane and N,N-dimethylformamide, more preferably acetone or N-methylpyrrolidone; the amount ratio of the binder and the second solvent is preferably (0.1-1) g:100 mL, more preferably (0.3-0.5) g:100 mL. In the present invention, the binder and the second solvent are preferably mixed by ultrasound, and the time of the ultrasound is based on mixing the binder. In the present invention, the binder can, on the one hand, enable the graphene and the electrode substrate to be firmly compounded, and on the other hand, can support the nanopores of the graphene to avoid damaging the graphene nanopores.

[0052] After obtaining the graphene dispersion and the binder solution, the present invention mixes and concentrates the graphene dispersion and the binder solution to obtain a graphene slurry. In the present invention, the volume ratio of the graphene dispersion and the binder solution is preferably 1:1 to 1.5. In the present invention, the method for mixing the graphene dispersion and the binder solution is preferably: adding the graphene dispersion to the binder solution, followed by stirring and ultrasonication; the stirring and ultrasonication time is based on the uniform mixing of the components. In the present invention, the concentration method is preferably a water bath method, an oil bath method, a rotary evaporation method, a suction filtration method, or a filtration method; the volume of the liquid after concentration is preferably 2 to 10% of the volume of the liquid before concentration, more preferably 2.5 to 5.5%. Through the concentration, the present invention obtains a graphene slurry with a suitable coating viscosity.

[0053] After obtaining the graphene slurry, the present invention coats the graphene slurry on the surface of a conductive substrate, and heat-treats the coated conductive substrate to obtain a conductive carrier. In the present invention, the material of the conductive substrate preferably includes titanium, nickel, zinc, steel, zinc, carbon, molybdenum, copper, gold, silver, platinum, palladium, indium tin oxide, fluorine-doped zinc oxide, antimony-doped tin oxide or aluminum-doped zinc oxide, and is more preferably titanium; the shape of the conductive substrate preferably includes a thin sheet, a coated flexible film, a block or a foam. Before the coating, the present invention also preferably subjects the conductive substrate to a hydrophilic treatment, and the hydrophilic treatment preferably includes one or more of a strong acid treatment, a strong base treatment, a hydrogen peroxide treatment and a plasma treatment; the hydrophilic treatment of the present invention facilitates the graphene slurry to extend and evenly cover the surface of the conductive substrate, and can also remove impurities on the surface of the battery substrate. In the present invention, the coating method preferably includes immersion, spraying or blade coating, more preferably blade coating; the coating is based on forming a uniform wet coating on the conductive substrate; the coating thickness is preferably 200 to 600 microns.

[0054] In the present invention, the heat treatment includes a first heat treatment and a second heat treatment in sequence; the temperature of the first heat treatment is 50-100°C, more preferably 80-100°C, and the temperature of the second heat treatment is preferably 300-500°C, more preferably 350-450°C; the time of the first heat treatment is preferably 1-2 hours, more preferably 1-1.5 hours, and the time of the second heat treatment is preferably 0.5-2 hours, more preferably 0.5-1 hour. In the present invention, the heat treatment is preferably carried out in a muffle furnace or a tube furnace. The present invention fully volatilizes the solvent and water through the first heat treatment, and partially decomposes the binder and volatilizes the co-solvent through the second heat treatment, leaving a co-plated layer of graphene and binder (the binder will occupy some pores of the graphene, but will not affect the electron conduction between the graphene particles). The setting of the heating program has a great influence on the composite of the graphene coating and the electrode substrate. If the first heat treatment is omitted and the second heat treatment is directly carried out, large bubbles will appear in the graphene coating, causing it to peel off easily. The present invention can fully eliminate defects such as bubbles in the graphene coating through step-by-step heat treatment, and improve the bonding strength between the graphene coating and the electrode substrate.

[0055] The preparation method provided by the present invention can firmly bond graphene to the electrode substrate, forming a strong graphene coating on the substrate surface, thus solving the problem of graphene easily peeling off the substrate. It can also fully support the graphene's nanoporous structure, introducing abundant nanopores and a superior specific surface area into the electrode substrate, thereby facilitating the dispersed and enhanced contact between the conductive carrier and the reaction solution, thereby improving the electron transfer rate. Furthermore, the method provided by the present invention is simple and easy to operate.

[0056] The fixed electrode provided by the present invention includes a ferredoxin-NADP+ reductase (FNR) and a reaction enzyme supported on the conductive carrier. The ferredoxin-NADP+ reductase and the reaction enzyme are supported in the pore structure of the porous graphene layer; the reaction enzyme is one or more of an oxidoreductase, a lyase, an isomerase, and a transferase.

[0057] In the present invention, the reaction enzyme is preferably an oxidoreductase, and more preferably glutamate dehydrogenase or alcohol dehydrogenase.

[0058] In the present invention, the mass ratio of the ferredoxin-NADP+ reductase to the reaction enzyme is preferably 1:(0.1-10).

[0059] In the present invention, the percentage of the mass of the ferredoxin-NADP+ reductase (FNR) to the total mass of the conductive carrier is preferably 0.1 to 0.5%.

[0060] Alternatively, in the present invention, the percentage of the mass of the reaction enzyme to the total mass of the conductive carrier is preferably 0.1-1%.

[0061] The present invention provides a method for preparing the immobilized enzyme electrode described in the above technical solution, comprising the following steps:

[0062] The ferredoxin-NADP+ reductase solution is coated on the surface of the conductive support (hereinafter referred to as the first coating), and a first loading electrode is obtained after a first standing.

[0063] The reaction enzyme solution is coated (hereinafter referred to as the second coating) on ​​the surface of the first load electrode, and the immobilized enzyme electrode is obtained after a second static state; the temperatures of the first static state and the second static state are independently ≤5°C.

[0064] In the present invention, a ferredoxin-NADP+ reductase solution is first coated on the surface of a conductive carrier, and then allowed to stand for a first time to obtain a first load electrode.

[0065] In the present invention, the ferredoxin-NADP+ reductase solution preferably includes FNR and a buffer solution. The buffer solution is a tris(hydroxymethyl)aminopropanesulfonic acid (TAPS) aqueous solution, the pH value of the TAPS aqueous solution preferably being 8-9, and the molar concentration of the TAPS aqueous solution preferably being 0.05 mol / L. The mass concentration of the ferredoxin-NADP+ reductase solution is preferably 10-20 mg / mL, more preferably 12-16 mg / mL.

[0066] The present invention has no special requirements for the specific implementation of the first coating. In the present invention, the specific implementation of the first coating is preferably drip coating. In the present invention, the temperature of the first static state is preferably 0-5°C. The holding time of the first static state is preferably 20-30 minutes, more preferably 25 minutes.

[0067] After obtaining the first loading electrode, the present invention applies the reaction enzyme solution on the surface of the first loading electrode for a second time, and then allows the electrode to stand for a second time to obtain the immobilized enzyme electrode.

[0068] In the present invention, the reaction enzyme solution preferably comprises a reaction enzyme and a buffer solution. The buffer solution is a tris(hydroxymethyl)aminopropanesulfonic acid (TAPS) aqueous solution. The pH value of the TAPS aqueous solution is preferably 8-9, and the molar concentration of the TAPS aqueous solution is preferably 0.05 mol / L. The mass concentration of the reaction enzyme solution is preferably 5-15 mg / mL, more preferably 6-12 mg / mL.

[0069] The present invention has no particular requirements for the specific implementation of the second coating. In the present invention, the specific implementation of the second coating is preferably drop coating. In the present invention, the temperature of the second static state is preferably 0-5°C. The holding time of the second static state is preferably 20-30 minutes, more preferably 25 minutes.

[0070] The present invention provides the use of the immobilized enzyme electrode described in the above technical solution or the immobilized enzyme electrode prepared by the preparation method described in the above technical solution in electrocatalytic enzyme cascade reaction.

[0071] In the present invention, the use of the immobilized enzyme electrode in an electrocatalytic enzyme cascade reaction preferably includes: employing a three-electrode system, with the immobilized enzyme electrode as the working electrode, Ag / AgCl as the reference electrode, and a platinum sheet as the counter electrode, to conduct an enzyme-catalyzed reaction in an electrolytic reaction solution. The electrolytic reaction solution comprises an enzyme reaction substrate, NADP+, and a solvent. The solvent is preferably a TAPS aqueous solution. The pH of the TAPS aqueous solution is preferably 8-9, and the molar concentration of the TAPS aqueous solution is preferably 0.05 mol / L. The enzyme reaction substrate is preferably α-ketoglutaric acid or 4-phenyl-2-butanone; the molar concentration of the enzyme reaction substrate in the electrolytic reaction solution is preferably 6-8 mmol / L, and the molar concentration of NADP+ is preferably 20 μmol / L. In the present invention, the enzyme-catalyzed reaction is preferably a constant-voltage direct current electrolytic reduction reaction. In the present invention, the Ag / AgCl electrode is used as the reference electrode, and the voltage of the working electrode is preferably -0.75 to -0.9 V. In the present invention, the temperature of the electrolytic reduction reaction is preferably 25 to 50° C.; the time is preferably 12 to 24 hours; the pressure of the electrolytic reduction reaction is standard atmospheric pressure; and the pH value of the electrolytic reduction reaction is preferably 2 to 10.

[0072] In the present invention, the specific implementation method of the application of the fixed enzyme electrode in the electrocatalytic enzyme cascade reaction is preferably as follows: first, the reference electrode is placed in a NaCl solution, the working electrode and the counter electrode are placed in a blank HEPES buffer solution, the external power supply is turned on, the CA voltage is set, and the CA graph is scanned. After the baseline is balanced, the power supply is turned off; the HEPES buffer solution is replaced with a solution containing an enzyme reaction substrate (i.e., an electrolyte), the power supply is turned on again, the CA voltage is set, and the CA graph is scanned. After the baseline is balanced, NADP+ is added to the electrolyte, and the current is monitored at any time during the reaction.

[0073] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0074] Example 1

[0075] The preparation steps of the conductive carrier are as follows:

[0076] Prepare graphene dispersion: weigh 0.3 g of graphene powder (multilayer graphene), mix it with 0.1 mL of glacial acetic acid in a fume hood, add deionized water twice and mix evenly, each time adding 2 mL of deionized water, then add N-methylpyrrolidone four times and mix evenly, each time adding 4 mL of N-methylpyrrolidone, then add 70 mL of N-methylpyrrolidone, stir for 15 minutes, and then ultrasonicate for 30 minutes; then add 2 g of terpineol, stir for 20 minutes, and then ultrasonicate for 40 minutes to obtain a graphene dispersion.

[0077] Prepare the binder solution: weigh 0.5 g of ethyl cellulose and 130 mL of N-methylpyrrolidone and mix them by ultrasonication until they are uniformly mixed to obtain a binder solution.

[0078] Preparation of graphene slurry: Add the graphene dispersion into the binder solution, stir for 20 minutes, and then ultrasonicate for 40 minutes, and then concentrate to 10 mL in an 80°C constant temperature oil bath to obtain graphene slurry.

[0079] Graphene slurry coating: soak the titanium sheet in 1 mol / L HCl solution, heat treat at 50°C for 30 min, then rinse with deionized water and then rinse three times with ethanol; take 100 μL of graphene slurry and evenly coat it on the pretreated titanium substrate using a doctor blade coating method. The coating thickness is about 500 microns. Then, place the coated electrode material in a muffle furnace at a constant temperature of 100°C for 1 hour, heat to 450°C, and keep warm for 30 minutes to obtain a conductive carrier.

[0080] The physical photograph and electron microscope image of the conductive carrier prepared in Example 1 are as follows: Figure 1 and Figure 2 As shown. Figure 2 The electron microscope image shows that the flake-like graphene and the remaining clusters of ethyl cellulose together create micron- and nanometer-scale channels. The graphene ensures the conductivity of the coating, while the ethyl cellulose helps the graphene to be firmly bonded.

[0081] The preparation steps of the immobilized enzyme electrode are as follows:

[0082] 6 μL of FNR was mixed with 10 μL of pH 8 tris(hydroxymethyl)methylaminopropanesulfonic acid (0.05 mol / LTAPS) buffer solution to obtain an FNR solution with a FNR concentration of 10 mg / mL. The solution was evenly applied to the surface of the conductive support and placed on ice for 25 minutes to obtain the first loaded electrode.

[0083] 14 μL of glutamate dehydrogenase was mixed with 30 μL of TAPS buffer solution (0.05 mol / L) at pH 8 to obtain a glutamate dehydrogenase solution with a mass concentration of 15 mg / mL. The solution was evenly applied on the surface of the first load electrode and placed on ice for 25 minutes to obtain an immobilized enzyme electrode.

[0084] An electrolytic cell was assembled with the fixed enzyme electrode as the working electrode, the platinum sheet as the counter electrode, and the Ag / AgCl as the reference electrode. A blank pH 8 TAPS buffer solution (0.05 mol / L) was first placed in the electrolytic cell. The cell was connected to an Ivium electrochemical workstation, the voltage was set to -0.75 V, and the constant voltage was scanned for about 20 minutes. After the current stabilized, the blank pH 8 TAPS buffer solution (0.05 mol / L) was replaced with the reaction solution; the reaction solution was a pH 8 TAPS buffer solution containing the reaction substrate. 8 TAPS buffer solution (0.05mol / L), wherein the reaction substrate is α-ketoglutaric acid, the molar concentration of α-ketoglutaric acid is 6mmol / L, the reaction solution is stirred magnetically (800 rpm), the voltage is set to -0.75V, and the constant voltage scan is performed for about 20 minutes. After the current stabilizes again, a pH 8 TAPS buffer solution (0.05mol / L) containing NADP+ is added through a needle injection to make the molar concentration of NADP+ in the electrolyte 20μmol / L. The reaction is carried out at a voltage of -0.75V and room temperature for 12h. The curve of the change of current over time during the reaction is shown as follows: Figure 3 As shown. Figure 3 As can be seen in the figure, after NADP+ is added, the current begins to rapidly increase to -0.2 mA at 2 hours, indicating that the enzyme begins to reduce α-ketoglutarate. The current decreases over time, indicating that the reaction rate slows as the α-ketoglutarate concentration decreases. After 23 hours of reaction, the current levels off, indicating that the α-ketoglutarate is essentially consumed and the reaction has reached equilibrium.

[0085] The samples before reaction ( Figure 4 ) and after 12h of reaction ( Figure 5 ) H NMR spectrum. Figure 4 and Figure 5 The two hydrogen spectra of the reactants and products intercepted the characteristic 0-3.8ppm range. Figure 4 and Figure 5 The numbers 1, 2 and 3 in the figure respectively identify the peaks of the corresponding characteristic H atoms. Figure 4 and Figure 5 It can be seen that the hydrogen peaks at positions 1 and 2 of the product shift to the high field, indicating that the carbonyl group is reduced to the amino group. In addition, a new peak at 3.8 ppm appears, which comes from -NH3 + The results show that the carbonyl group is reduced to the amino group and α-ketoglutarate is reduced to L-glutamate.

[0086] Example 2

[0087] A conductive support was prepared according to the method of Example 1.

[0088] The preparation steps of the immobilized enzyme electrode are as follows:

[0089] 15 μL of FNR was mixed with 35 μL of pH 9 tris(hydroxymethyl)methylaminopropanesulfonic acid (0.05 mol / LTAPS) buffer solution to obtain an FNR solution with a FNR concentration of 20 mg / mL. The solution was evenly applied to the surface of the conductive support and placed on ice for 25 minutes to obtain the first loaded electrode.

[0090] 4 μL of alcohol dehydrogenase was mixed with 16 μL of TAPS buffer solution (0.05 mol / L) at pH 9 to obtain an alcohol dehydrogenase solution with a mass concentration of 5 mg / mL. The solution was evenly applied on the surface of the first load electrode and placed on ice for 25 minutes to obtain an immobilized enzyme electrode.

[0091] An electrolytic cell was assembled with the fixed enzyme electrode as the working electrode, the platinum sheet as the counter electrode, and the Ag / AgCl as the reference electrode. A blank pH 9 TAPS buffer solution (0.05 mol / L) was first placed in the electrolytic cell. The cell was connected to an Ivium electrochemical workstation, the voltage was set to -0.75 V, and the constant voltage was scanned for about 20 minutes. After the current stabilized, the blank pH 9 TAPS buffer solution (0.05 mol / L) was replaced with the reaction solution; the reaction solution was a pH 9 TAPS buffer solution containing the reaction substrate. APS buffer solution (0.05mol / L), in which the reaction substrate is 4-phenyl-2-butanone, the molar concentration of 4-phenyl-2-butanone is 8mmol / L, the reaction solution is stirred magnetically (800 rpm), the voltage is set to -0.9V, and the constant voltage scan is performed for about 20 minutes. After the current stabilizes again, a pH 8 TAPS buffer solution (0.05mol / L) containing NADP+ is added through a needle injection to make the molar concentration of NADP+ in the electrolyte 20μmol / L. The reaction is carried out at a voltage of -0.9V and room temperature for 18h. The curve of the change of current over time during the reaction is shown as follows: Figure 6 shown. Figure 6 The figure shows the curve of current changing with time. The magnitude of the current reflects the rate of reaction. The more negative the current, the greater the Faraday current generated by the reduction product. Figure 6As can be seen in the figure, at 0.5 h after the addition of NADP+, the current rapidly increases to -0.24 mA, indicating that the enzyme-catalyzed reduction of 4-phenyl-2-butanone begins. The current decreases over time, indicating that the reaction rate slows as the 4-phenyl-2-butanone concentration decreases. After 16 h of reaction, the current levels off, indicating that the 4-phenyl-2-butanone is essentially consumed and the reaction has reached equilibrium.

[0092] The samples before reaction ( Figure 7 ) and after 18h of reaction ( Figure 8 ) H NMR spectrum. Figure 7 and Figure 8 The two hydrogen spectra of the reactants and products intercepted the characteristic 0-3.5ppm range. Figure 4 and Figure 5 The numbers 1 and 2 in the figure respectively identify the peaks of the corresponding characteristic H atoms. Figure 7 and Figure 8 , it can be seen that the hydrogen peak of the product shifts to the high field, indicating that the carbonyl group is reduced to alcohol, that is, 4-phenyl-2-butanone is reduced to 4-phenyl-2-butanol.

[0093] Example 3

[0094] The preparation method and reaction conditions of the immobilized enzyme electrode are basically the same as those of Example 2, except that the reaction temperature is increased from room temperature to 50°C. Figure 9 It can be seen that at 1h, after adding NADP+, the current began to increase rapidly to -0.19mA. Figure 6 This shows that under the same reaction conditions and enzyme loading, the enzyme activity can still be maintained at 80% of that at room temperature under the reaction conditions of 50℃.

[0095] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An immobilized enzyme electrode, characterized in that The invention comprises a conductive carrier and a ferredoxin-NADP+ reductase and a reaction enzyme loaded on the conductive carrier, wherein the reaction enzyme is glutamate dehydrogenase or alcohol dehydrogenase; the conductive carrier comprises a conductive substrate and a porous graphene layer arranged on the surface of the conductive substrate, wherein the pore structure of the porous graphene layer is nanopores; the ferredoxin-NADP+ reductase and the reaction enzyme are loaded in the pore structure of the porous graphene layer; The method for preparing the conductive carrier comprises the following steps: mixing graphene, glacial acetic acid, water, N-methylpyrrolidone and terpineol to obtain a graphene dispersion; mixing ethyl cellulose and N-methyl pyrrolidone to obtain a binder solution; The graphene dispersion and the binder solution are mixed and concentrated to obtain a graphene slurry; coating the graphene slurry on a surface of a conductive substrate, and heat-treating the coated conductive substrate to obtain a conductive carrier; The heat treatment includes a first heat treatment and a second heat treatment performed sequentially, wherein the temperature of the first heat treatment is 100° C. and the time of the first heat treatment is 1 hour, and the temperature of the second heat treatment is 450° C. and the time of the second heat treatment is 0.5 hour.

2. The immobilized enzyme electrode according to claim 1, characterized in that The mass ratio of the ferredoxin-NADP+ reductase to the reaction enzyme is 1:(0.1-10).

3. The method for preparing the immobilized enzyme electrode according to claim 1 or 2, characterized in that: The following steps are involved: The ferredoxin-NADP+ reductase solution is coated on the surface of the conductive support and allowed to stand for a first time to obtain a first loaded electrode; The reaction enzyme solution is coated on the surface of the first load electrode, and the immobilized enzyme electrode is obtained after a second static state; the temperature of the first static state and the second static state are independently ≤5°C.

4. The preparation method according to claim 3, characterized in that The holding time of the first standing and the second standing are independently 20 to 30 minutes.

5. The preparation method according to claim 3, characterized in that The mass concentration of the ferredoxin-NADP+ reductase solution is 10-20 mg / mL.

6. The preparation method according to claim 3, characterized in that The mass concentration of the reaction enzyme solution is 5-15 mg / mL.

7. Use of the immobilized enzyme electrode according to claim 1 or 2 or the immobilized enzyme electrode prepared by the preparation method according to any one of claims 3 to 6 in an electrocatalytic enzyme cascade reaction.

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