Redox enzyme electrode for enzyme electrocatalytic reduction and preparation method thereof and enzyme electroreactor thereof

Abstract

The present invention relates to an oxidoreductase electrode for enzyme electrocatalytic reduction, a preparation method thereof and an enzyme electrocatalytic reactor thereof. Compared with the requirements of low detection limit and high sensitivity of enzyme electrode detection, enzyme electrocatalysis requires tolerance to high-concentration substrates, good enzyme stability and high catalytic efficiency. The coenzyme of the present invention is co-immobilized with the enzyme on the electrode, which can realize in-situ regeneration of the coenzyme, improve reaction efficiency, realize enzyme electrocatalytic efficient preparation of chiral compounds, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biocatalysis technology, and in particular to a high-efficiency enzyme bioelectrocatalytic reactor and a method and application thereof for reducing and preparing chiral compounds. Background Art

[0002] Bioelectrocatalytic synthesis has no by-products and high selectivity, and has attracted attention for its advantages such as high efficiency and green environmental protection. Enzymes can realize a variety of electrocatalytic processes through bidirectional electron transfer and energy metabolism with the external environment. At present, bioelectrocatalysis is mainly concentrated in microbial electrosynthesis systems, while enzyme electrocatalytic reduction is less and focuses on the fields of catalytic immobilization of CO2 and nitrogenase by multi-enzyme coupled electrocatalytic systems. However, there are few studies on the asymmetric electroreduction of high value-added chiral compounds, such as non-natural chiral amino acids, chiral alcohols, and chiral alcohols. Compared with the requirements of low detection limit and high sensitivity of enzyme electrode detection, enzyme electrocatalysis requires tolerance to high concentration substrates, good enzyme stability, high catalytic efficiency, focusing on catalytic preparation, and requiring efficient regeneration of coenzymes. However, the bottleneck problems of bioelectrocatalytic asymmetric reduction of dehydrogenases that rely on NAD(P)H include not only the difficulty of coenzyme regeneration, but also the poor stability of enzymes on the electrode surface, limited enzyme loading, and difficulty in large-scale amplification. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a redox enzyme electroreactor and a preparation method and application thereof. The present invention constructs a new type of redox enzyme electroreactor, which provides a good microenvironment for the electrochemical reaction of biomolecules, has high enzyme electrocatalytic reduction efficiency and good stability, and can be used for bioelectrocatalytic preparation of chiral drug intermediates.

[0004] One of the technical solutions adopted by the present invention to solve the technical problem is:

[0005] A redox enzyme electrode for enzyme electrocatalytic reduction comprises a substrate electrode, an electron conductor, an electron mediator, an enzyme and a coenzyme; the electron conductor comprises a combination of at least two of graphene oxide (GO), reduced graphene oxide (rGO), carbon nanotubes (MWNT), polydopamine (PDA), nanogold, MOF, polyethyleneimine (PEI) or biomimetic mineralized TiO2 materials; the coenzyme comprises at least one of ionic coenzymes NADH-IL or PEI-Fc-NADH; the coenzyme and the enzyme are co-fixed on the enzyme electrode.

[0006] Wherein, the enzyme is a NADH-dependent or NADPH-dependent dehydrogenase, preferably including at least one of amino acid dehydrogenase, amine dehydrogenase, alcohol dehydrogenase or ketoreductase.

[0007] Wherein, the substrate electrode includes a carbon paper electrode (CP), a glassy carbon electrode (GCE), or a graphite electrode, etc., preferably a substrate electrode with a larger specific surface area, such as a carbon paper electrode and a pyrolytic graphite electrode.

[0008] Wherein, the electron mediator includes at least one of 5-methylphenazine methyl sulfate, toluidine blue, Meldola blue or methylene blue.

[0009] Among them, the electron conductor can be the following combination: the electron conductor includes GO and MOF; or the electron conductor includes carbon nanotubes, PDA and nanogold; or the electron conductor includes rGO, PDA and nanogold; or the electron conductor includes rGO, PEI and biomimetic mineralized TiO2 material.

[0010] In the redox enzyme electrode of the present invention, the coenzyme and the enzyme are co-immobilized on the electrode to achieve in-situ regeneration of the coenzyme and improve the catalytic efficiency. Ionic liquids can be used as modifiers and adhesives for traditional glassy carbon electrodes. The graphene with the addition of ILs can solve the problem that it is difficult to directly modify the electrode surface, and the higher conductivity of IL accelerates the electron transfer between the coenzyme NAD(P)H and the electrode. The substrate electrode with a large specific surface area such as carbon paper increases the enzyme loading on the electrode surface. The redox mediator organic small molecules act as a "molecular bridge" between the electrochemically active protein and the inorganic material electrode, accelerate electron shuttling, and improve the biocompatibility of the electrode material.

[0011] The second technical solution adopted by the present invention to solve its technical problem is:

[0012] A method for preparing a redox enzyme electrode comprises compounding an immobilized mixture of an enzyme and an electron conductor with an electron mediator and a coenzyme on the surface of a substrate electrode to obtain the redox enzyme electrode.

[0013] Specifically, the method for preparing the immobilized mixture of enzyme and electron conductor comprises:

[0014] When the electron conductor includes GO and MOF, metal ions of MOF are added to the graphene oxide solution, and then 0.1-100 mg / mL of the enzyme is added, and a ligand of MOF is added under stirring at 0-50° C. to react to obtain a metal-organic framework (MOF) and immobilize the enzyme in situ to obtain an immobilized mixture of the enzyme and the electron conductor; further, the reaction of the in situ immobilized enzyme can be carried out on a substrate electrode; or,

[0015] When the electron conductor includes carbon nanotubes, PDA and nanogold, polydopamine is added to the carbon nanotube solution to obtain a carbon nanotube-polydopamine solution; the carbon nanotube-polydopamine solution is reacted with a nanogold precursor to obtain a carbon nanotube-polydopamine solution containing nanogold, and the enzyme is mixed with the carbon nanotube-polydopamine solution containing nanogold to obtain an immobilized mixture of the enzyme and the electron conductor; or,

[0016] When the electron conductor includes rGO, PDA and nano-gold, DA and the enzyme are added to the rGO solution to react with the nano-gold precursor and immobilize the enzyme to obtain an immobilized mixture of the enzyme and the electron conductor; or,

[0017] When the electronic conductor includes rGO, PEI and biomimetic mineralized TiO2 material, the enzyme is coordinated and fixed on reduced graphene oxide-polyethyleneimine (rGO-PEI), and then Ti-BALDH is added, and TiO2 biomimetic mineralization is induced by PEI to immobilize the enzyme, thereby obtaining an immobilized mixture of the enzyme and the electronic conductor. Further, the enzyme is coordinated and fixed on rGO-PEI, and then Ti-BALDH, an electronic mediator, and a coenzyme are added, and TiO2 biomimetic mineralization is induced by PEI to simultaneously immobilize the enzyme and the coenzyme. The process of biomimetic mineralization to immobilize the enzyme or to simultaneously immobilize the enzyme and the coenzyme can be carried out on the substrate electrode.

[0018] Further, the ligand of the metal-organic framework (MOF) includes one of trimesic acid (H3BTC; benzene-1,3,5-tricarboxylic acid), 2,3,6,7,10,11-hexaaminotriphenyl hexahydrochloride (HITP), 2,3,6,7,10,11-hexahydroxytriphenyl (HHTP), hexaaminobenzene (3 hydrochloride) (HAB), Nu-1006 or Nu-1007; the metal ion includes Co 2+ , Cu 2+ 、Zn 2+ 、Ni 2+ or Zr 4+ At least one of (CoCl2, CuCl2, ZnCl2, NiCl2, ZrCl4, etc.). The formed MOF includes Ni3(HITP)2, Co-HAB, Cu3(HHTP)2, Ni-CAT, etc.

[0019] Furthermore, the ionic coenzyme NADH-IL or polymer-modified coenzyme (PEI-Fc-NADH) coenzyme forms a gel with the electron mediator, and the immobilized mixture of the enzyme and the electron conductor is compounded with the gel on the surface of the substrate electrode, so that the dehydrogenase, coenzyme and electron mediator are co-fixed on the electrode surface, the coenzyme is regenerated in situ, and the catalytic efficiency is improved.

[0020] Furthermore, the electron mediator, the coenzyme (NADH-IL or PEI-Fc-NADH) and the ionic liquid (for dissolving NADH-IL or PEI-Fc-NADH) form the gel; the ionic liquid includes at least one of 1-ethyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium chloride, or N,N'-(methylene)bis(1-(3-vinylimidazole))bromide.

[0021] Furthermore, the preparation method of the PEI-Fc-NADH (polymer covalently linked coenzyme) includes: preparing 0.1-0.7 mmol / mL and 0.05-0.2 mmol / mL ethanol solutions of polyethyleneimine (PEI) and ferrocenecarboxaldehyde (Fc), respectively, adding the ethanol solution of Fc dropwise to the ethanol solution of PEI within 1-3 hours, and continuing to stir and react for 1-3 hours. Add sodium borohydride with a final concentration of 0.1-1.0 mmol / mL, and continue to react for 1-4 hours. Add 1-10 mL of distilled water to the residue, dialyze with distilled water for 12 hours to obtain PEI-Fc. Dissolve 10-100 mmol of succinic anhydride in 6-30 mL of dimethyl sulfoxide, and add 0.0745-0.5 g of NAD + Add to the DMSO solution of succinic anhydride and place at room temperature to react for 12 hours. Add 10-30 mL of acetone to the mixed solution, centrifuge to obtain a precipitate, add 2-5 mL of phosphate buffer solution with pH = 4-6 containing 1-3 mmol EDC, and activate the hydroxyl group with EDC for 1-4 hours. After activation, add 5-10 mL of PEI-Fc (pH = 4-6) to the above solution, adjust pH = 4.7, and react at 0-4°C for 12-24 hours. After the reaction is completed, dialyze with distilled water for 12 hours to obtain PEI-Fc-NADH.

[0022] The third technical solution adopted by the present invention to solve its technical problem is:

[0023] An enzyme electroreactor for enzyme electrocatalytic reduction, the enzyme electroreactor adopts a three-electrode system, with the above-mentioned redox enzyme electrode as the working electrode; the reference electrode is selected from a saturated calomel electrode, a hydrogen electrode, a silver|silver chloride electrode or a mercury|mercury oxide electrode; the counter electrode is a platinum wire electrode or a carbon electrode.

[0024] The fourth technical solution adopted by the present invention to solve its technical problem is:

[0025] A method for catalytic reduction reaction using an enzyme-electroreactor, wherein the reaction is carried out in the manner of cyclic voltammetry and / or chronoamperometry; the potential scanning rate of the cyclic voltammetry is 1 to 500 mV / s; the reaction is carried out in a buffer solution to which 1 to 20 mg of coenzyme NADH is added, and N2 is introduced before the reaction; the reaction substrates are ketoacid, a substrate of amino acid dehydrogenase, ketone, a substrate of amine dehydrogenase, and ketone, a substrate of alcohol dehydrogenase; the concentration range of the substrates is 5 mM to 500 mM; the buffer solution is at least one of phosphate buffer, borate buffer, citrate buffer, carbonate buffer, Tris-HCl buffer, etc.

[0026] Specifically, a 1.5-100 mL bioelectrocatalytic microreactor was designed based on a disposable plastic test tube; a three-electrode system of the reactor was designed, with carbon paper (5×5 mm~5×5 cm) fixed with enzymes and gel as the working electrode, a platinum wire electrode as the counter electrode, and Ag / AgCl as the reference electrode. The reaction was carried out in 0.5-100 mL phosphate buffer (1-500 mM, pH 5-13) with NADH-IL (NADH-ionic liquid). N2 was introduced before the reaction. Cyclic voltammetry was used during the test, in which no substrate was added to the blank control. The scan rate of the cyclic voltammetry test was 50 mV / s, the scan range was -1 V to 1 V, and it was carried out in a buffer solution with a pH of 7.0. The time of the chronoamperometric current test was 30-1800 s, and the substrate was added every 30 s. The current density was adjusted to 0.1-1.5 mA / cm 2 Calculate the total transformation number (TTN) of coenzyme regeneration and optimize the electrochemical regeneration conditions. The voltage is 1-200mV / s, the scanning range is -1V-1V, and it is carried out in a buffer solution with a pH of 6.0-12.0. The time of the chronoamperometric current test is 30-1800s, 1200s, and the substrate is added once at 1mM-500mM. The current density is regulated at 0.1-1.5mA / cm 2 .

[0027] Unless otherwise specified, the equipment, reagents, processes, parameters, etc. involved in the present invention are conventional equipment, reagents, processes, parameters, etc. and are not given as embodiments.

[0028] All ranges recited herein include all point values ​​within the range.

[0029] The terms “approximately”, “about” or “around” as used herein refer to within ±20% of the range or value.

[0030] In the present invention, unless otherwise specified or having common meanings in the field, % refers to mass percentage and ratio refers to mass ratio.

[0031] In the present invention, the "room temperature" refers to the normal ambient temperature, which may be 10 to 30°C.

[0032] The beneficial effects of the present invention are as follows:

[0033] 1. The present invention uses carbon paper, graphene oxide (GO), reduced graphene oxide (rGO), carbon nanotubes (CNT) and other carbon nanomaterials and conductive polymers, ionic liquids and the like as electron transfer materials for enzyme electrode preparation, and achieves stable enzyme immobilization and efficient electron transfer with superior electronic properties, chemically modifiable properties and good biocompatibility. The enzyme is immobilized in a directional manner using functional electrode materials with good biocompatibility, achieving in-situ regeneration of coenzymes, increasing the loading amount of enzyme electrodes, and improving enzyme stability, and has the advantages of simple preparation method, low cost, good stability and repeatability.

[0034] 2. Amino acid dehydrogenase (AaDH), alcohol dehydrogenase and amine dehydrogenase are important enzymes in biological metabolic pathways. AaDH uses NH3 as an amino donor to catalyze the asymmetric reduction amination reaction of keto acids, and can synthesize a series of chiral natural amino acids, unnatural amino acids and chiral amines, etc., which are important chiral building blocks for fine chemicals such as medicines. The enzyme-electroreactor of the present invention can successfully catalyze the asymmetric electroreduction of various dehydrogenases and efficiently obtain a variety of chiral compounds, and has good application prospects in the preparation of chiral drug intermediates and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0036] Figure 1 Schematic diagram of the reactor structure in Example 1 of the present invention (left) and a photo of the actual reactor (right).

[0037] Figure 2 This is a picture of the enzyme immobilized on carbon paper in Example 1 of the present invention.

[0038] Figure 3 This is an electron microscope image of the immobilized enzyme CP / Cu3(BTC)2 / GO-AaDH obtained in Example 1 of the present invention.

[0039] Figure 4 This is the coenzyme regeneration curve in Example 1 of the present invention.

[0040] Figure 5 This is a photo of the enzyme electrocatalytic reactor in Example 3 of the present invention.

[0041] Figure 6 This is the SEM characterization of MWNTs-PDA in Example 3 of the present invention. DETAILED DESCRIPTION

[0042] The present invention is described in detail below by way of examples.

[0043] Example 1

[0044] (1) Preparation of crude amino acid dehydrogenase: Based on the original sequence of amino acid dehydrogenase (AaDH) as shown in SEQ ID No. 1, the gene sequence of amino acid dehydrogenase was synthesized by a commercial company (Shanghai Biotechnology Co., Ltd.) using the PCR method and connected to the vector pET28a to construct a plasmid.

[0045] Connection of PCR product (gene sequence of amino acid dehydrogenase) and vector pET28a: Double digest the PCR product and vector pET28a with NdeI and XhoI, and react overnight at 4°C under the action of T4 DNA Ligase after purification to obtain the transformation plasmid. Competent cells were prepared using the Competent Cel Preparation Kit. Add 10 ng of transformation plasmid to the competent cells, mix gently, place on ice for 30 minutes, place in a 42°C water bath for 45 seconds, and immediately place on ice for 1 to 2 minutes. Add LB medium preheated at 37°C, shake and culture at 37°C for 1 hour, take an appropriate amount of bacterial solution to apply to LB solid plates with kanamycin resistance, and pick out single colonies after overnight culture at 37°C, which is the recombinant Escherichia coli (E.coli BL21(DE3) / pET28a).

[0046] Cultivation of recombinant E. coli BL21 (DE3) / pET28a: Inoculate the strain into 200 mL LB medium at a 1% inoculum. The composition of LB medium is 10.0 g / L tryptone, 5.0 g / L yeast powder, 10 g / L NaCl. The culture conditions are: initial pH 7.0, liquid volume fraction is 10%, culture temperature 37 ° C, shaker speed 200 rpm, culture time 6 hours. Add the inducer IPTG to a final concentration of 10 mg / mL, and continue to culture at 30 ° C, 200 rpm for 12 hours.

[0047] Preparation of crude enzyme solution: The fermentation liquid obtained after the cultivation was centrifuged in a refrigerated centrifuge (4°C, 8000rpm, 15min) to obtain cells, discard the supernatant, resuspend the precipitate with Tris-HCl buffer (pH 7.5), wash it thoroughly and centrifuge it, repeat the operation 3 times to obtain cells. The cell liquid was treated with an ultrasonic disruptor, and the probe of the cell disruptor was placed 1cm below the liquid surface. The disruption conditions were ultrasonic for 2 seconds, 4 seconds interval, 40 ultrasonic times, and a power of 200W. Then centrifuge at 4°C and 12,000rpm for 15min to remove insoluble cell debris, and the supernatant was the crude amino acid dehydrogenase enzyme solution.

[0048] (2) Preparation of pure amino acid dehydrogenase enzyme: The crude amino acid dehydrogenase enzyme solution was separated and purified using GE's His Trap nickel column, and ultrafiltration and desalting were performed using PALL's 10K ultrafiltration centrifuge tube. The purification column used in the purification process is a HisTrap HP column that can specifically purify proteins with His-tagged tags, and the steps include equilibration, loading, equilibration, elution, and column regeneration; the eluted part is collected and desalted using an ultrafiltration centrifuge tube; the liquid obtained after desalting is the pure amino acid dehydrogenase enzyme (AaDH with His-tag) liquid. Tris-HCl (0.05M, pH7.0) buffer is added thereto to prepare an aqueous solution containing amino acid dehydrogenase, and the amino acid dehydrogenase concentration therein is adjusted to 0.05-50 mg / mL as needed for standby use.

[0049] (3) Synthesis of ionic coenzyme NADH-IL: An ionic coenzyme in the form of an ionic liquid with nicotinamide coenzyme NADH as anion and different imidazoles as cations was designed and synthesized. N-methylimidazole (33 mL, 0.31 M) and n-butyl chloride (20 mL, 0.25 M) were added at a molar ratio of about 1.2:1. Under nitrogen protection and magnetic stirring, reflux was performed at 90 °C for 24 h to prepare 1-butyl-3-methylimidazole chloride and purify it. The 1-butyl-3-methylimidazole chloride ionic liquid was dissolved in water and slowly passed through a column filled with 717 anion exchange resin (the anion of 717 anion exchange resin before use was Cl - ), add an equal volume of equimolar NADH aqueous solution, stir at room temperature for 48 hours, purify and vacuum dry to obtain ionic coenzyme NADH-IL. NADH-IL NMR characterization: 1 H NMR(400Hz,D2O)δ=0.86(t,3H),1.27(m,2H),1.75(m,2H),3.84(s,3H),4.12(t,2H),4.21_4.71(m,15H),5.92(d,1H),5.97(d,1H), 7.26(s,1H),7.31(s,1H),8.05(s,1H),8.12(m,1H),8.34(s,1H),8.55(s,1H),8.73(s,1H),8.76(s,1H),9.09(d,1H),9.24(s,1H).

[0050] (4) Enzyme immobilization on carbon cloth: The fuel cell-specific carbon paper TGP-H-060 produced by Toray Industries, Japan, was used as a carrier. Graphene oxide was prepared by the Hummer method, and a uniformly dispersed GO solution was obtained after ultrasonic dispersion for 3 hours. 50mM and 15mM Cu(NO3)2·3H2O aqueous solutions and 25mM and 8mM H3BTC ethanol solutions were prepared respectively. The GO solution, Cu(NO3)2 aqueous solution and H3BTC ethanol solution were mixed and the AaDH solution with His-tag obtained in step (2) was added thereto (specifically, for example, Cu(NO3)2 aqueous solution was added to the GO solution, and the AaDH solution with His-tag obtained in step (2) was added thereto, and finally the ligand H3BTC ethanol solution was added thereto). The carbon paper TGP-H-060 was immersed in the above-mentioned mixed solution, and the CP / Cu3(BTC)2 / GO-AaDH was obtained by in-situ assembly driven by electrostatic action and metal coordination, as shown in FIG. Figure 3 ; Take the gel containing methylene green, NADH-IL and [EMIM]BF4 and drop it on the surface of the above CP / Cu3(BTC)2 / GO-AaDH, dry it at 4°C, and obtain a working modified electrode, such as Figure 2 Cu3(BTC)2 particles adsorbed on GO sheets have excellent adhesion and are then fixed on carbon paper to prepare three-dimensional electrode materials. The loading amount is 20 mg enzyme per 1 g of composite material.

[0051] (5) Design and performance characterization of bioelectrocatalytic reactor: A 50 mL bioelectrocatalytic microreactor was designed based on a disposable plastic test tube. The reactor was designed with a three-electrode system. The carbon paper (5×5 cm) immobilized with enzyme and gel prepared in step (4) was used as the working electrode (WE), the counter electrode (CE) was a platinum wire electrode, and Ag / AgCl was used as the reference electrode (RE). Figure 1 The reaction was carried out in 10 mL of phosphate buffer (0.02 mM, pH 7.4) with NADH-IL added. N2 was introduced before the reaction. Cyclic voltammetry was used during the test, and no substrate was added as a blank control. The cyclic voltammetry test was carried out at a scan rate of 50 mV / s and a scan range of -1 V to 1 V in a buffer solution with a pH of 8.0. The substrate phenylpyruvic acid was added at a concentration of 100 mM. The current density was regulated to 0.8 mA / cm 2 , reacted for 8h, and obtained an L-phenylalanine concentration of 85mM. The coenzyme regeneration performance test of the obtained enzyme bioelectroreactor is as follows Figure 4 shown.

[0052] Example 2

[0053] (1)~(2) As in Example 1.

[0054] (3) Preparation of polymer covalently linked coenzyme (PEI-Fc-NADH): Prepare 0.7 mmol / mL and 0.2 mmol / mL ethanol solutions of polyethyleneimine (PEI) and ferrocenecarboxaldehyde (Fc), respectively. Add the ethanol solution of Fc dropwise to the ethanol solution of PEI within 3 hours, and continue to stir and react for 3 hours. Add sodium borohydride to a final concentration of 1.0 mmol / mL and continue the reaction for 4 hours. Separate the solid and liquid, add 10 mL of distilled water to the residue, and dialyze with distilled water for 12 hours to obtain PEI-Fc. Dissolve 100 mmol of succinic anhydride in 30 mL of dimethyl sulfoxide, and add 0.5 g of NAD + Add to the DMSO solution of succinic anhydride and place at room temperature for reaction for 12 hours. Add 30 mL of acetone to the mixed solution, centrifuge to obtain a precipitate, add 5 mL of pH=6 phosphate buffer solution containing 3 mmol EDC, and activate the hydroxyl group with EDC for 4 hours. After activation, add 10 mL of PEI-Fc (pH=6) to the above solution, adjust pH=4.7, and react at 4°C for 24 hours. After the reaction is completed, dialyze with distilled water for 12 hours to obtain PEI-Fc-NADH.

[0055] (4) Enzyme immobilization on carbon cloth: Toray's fuel cell-specific carbon paper TGP-H-060 was used as a carrier. Graphene oxide was prepared by the Hummer method, and a uniformly dispersed GO solution was obtained after ultrasonic dispersion for 3 h. 50 mM and 15 mM Cu(NO3)2·3H2O aqueous solutions and 25 mM and 8 mM H3BTC ethanol solutions were prepared respectively. Cu(NO3)2 aqueous solution was added to the GO solution, and the AaDH solution with His-tag obtained in step (2) was added thereto, and finally the ligand H3BTC ethanol solution was added; the carbon paper TGP-H-060 was immersed in the above mixed solution, and CP / Cu3(BTC)2 / GO-AaDH was obtained by in-situ assembly driven by electrostatic action and metal coordination; a gel containing methylene green, PEI-Fc-NADH and [EMIM]BF4 was dropped on the surface of CP / Cu3(BTC)2 / GO-AaDH and dried at 4°C to obtain a working modified electrode.

[0056] (5) Design and performance characterization of bioelectrocatalytic reactor: A 50 mL bioelectrocatalytic microreactor was designed based on a disposable plastic test tube. The reactor was designed with a three-electrode system. The carbon paper (5×5 cm) immobilized with enzyme and PEI-Fc-NADH obtained in step (4) was used as the working electrode, the counter electrode was a platinum wire electrode, and Ag / AgCl was used as the reference electrode. The reaction was carried out in 10 mL phosphate buffer (0.02 mM, pH 7.4) with NADH-IL added. N2 was introduced before the reaction. Cyclic voltammetry was used during the test, and no substrate was added to the blank control. The scan rate of the cyclic voltammetry test was 50 mV / s, the scan range was -1 V to 1 V, and it was carried out in a buffer solution with a pH of 8.0. The substrate phenylpyruvic acid was added at a concentration of 100 mM. The current density was adjusted to 0.8 mA / cm 2 , react for 8 hours, and obtain an L-phenylalanine concentration of 85mM.

[0057] Example 3

[0058] (1) Preparation of crude amine dehydrogenase: Based on the original sequence of amine dehydrogenase, a commercial company (Shanghai Bioengineering Co., Ltd.) used PCR to synthesize the gene sequence of amine dehydrogenase as shown in SEQ ID NO. 2, which was used to connect with vector pET28a to construct a plasmid.

[0059] Connection of PCR product (gene sequence of amine dehydrogenase) and vector pET28a: Double digest the PCR product and vector pET28a with NdeI and XhoI, and react overnight at 4°C under the action of T4 DNA ligase after purification to obtain the transformation plasmid. Competent cells were prepared using the Competent Cell Preparation Kit. Add 10 ng of transformation plasmid to the competent cells, mix gently, place on ice for 30 minutes, place in a 42°C water bath for 45 seconds, and immediately place on ice for 1 to 2 minutes. Add LB medium preheated at 37°C, shake and culture at 37°C for 1 hour, take an appropriate amount of bacterial solution to apply to LB solid plates with kanamycin resistance, and pick out single colonies after overnight culture at 37°C, which is the recombinant Escherichia coli (E.coli BL21(DE3) / pET28a).

[0060] Cultivation of recombinant E. coli BL21 (DE3) / pET28a: Inoculate the strain into 200 mL LB medium at a 1% inoculum. The composition of LB medium is 10.0 g / L tryptone, 5.0 g / L yeast powder, 10 g / L NaCl. The culture conditions are: initial pH 7.0, liquid volume fraction is 10%, culture temperature 37 ° C, shaker speed 200 rpm, culture time 6 hours. Add the inducer IPTG to a final concentration of 10 mg / mL, and continue to culture at 30 ° C, 200 rpm for 2 hours.

[0061] Preparation of crude enzyme solution: The fermentation liquid obtained after the cultivation was centrifuged in a refrigerated centrifuge (4°C, 8000rpm, 15min) to obtain cells, discard the supernatant, resuspend the precipitate with Tris-HCl buffer (pH 7.5), wash it thoroughly and centrifuge it, repeat the operation 3 times to obtain cells. The cell liquid was treated with an ultrasonic disruptor, and the probe of the cell disruptor was placed 1cm below the liquid surface. The disruption conditions were ultrasonic for 2 seconds, 4 seconds interval, 40 ultrasonic times, and a power of 200W. Then centrifuge at 4°C and 12,000rpm for 15min to remove insoluble cell debris, and the supernatant was the crude amine dehydrogenase enzyme solution.

[0062] (2) Preparation of pure amine dehydrogenase enzyme: The crude amine dehydrogenase enzyme solution was separated and purified using a His Trap nickel column from GE, and ultrafiltration and desalting were performed using a 10K ultrafiltration centrifuge tube from PALL. The purification column used in the purification process is a HisTrap HP column that can specifically purify proteins with a His-tagged tag, and the steps include equilibration, loading, equilibration, elution, and column regeneration; the eluted portion was collected and desalted using an ultrafiltration centrifuge tube; the liquid obtained after desalting is the pure amine dehydrogenase enzyme liquid. Tris-HCl (0.05M, pH 7.0) buffer was added thereto to prepare an amine dehydrogenase solution containing amine dehydrogenase, and the amine dehydrogenase concentration therein was adjusted to 0.05-50 mg / mL for standby use as needed.

[0063] (3) Preparation of immobilized enzyme material: 20 mg of multi-walled carbon nanotubes (MWNTs) (Nanjing Jicang Nanotechnology Co., Ltd., multi-walled carbon nanotubes JCMT-95-11-10) were added to 20 mL of water and ultrasonically dispersed for 3 h to obtain a uniformly dispersed 1 mg / mL MWNTs solution. 40 mg of PDA was added under stirring and stirred at room temperature for 12 h. After centrifugation and resuspension, a 1 mg / mL MWNTs-PDA solution was prepared. The SEM image of MWNTs-PDA is shown in the figure. Figure 6 The MWNTs-PDA solution was mixed with the NaAuCl4 solution to obtain the MWNTs-PDA solution containing AuNPs.

[0064] (4) Preparation of MWNTs-PDA-AuNPs-AmDH: The amine dehydrogenase solution with an enzyme concentration of 5 mg / mL obtained in step (2) was mixed with the MWNTs-PDA solution containing AuNPs in a ratio of 2:1:1 at 4°C, centrifuged at 8000 rpm and the supernatant discarded to precipitate the amine dehydrogenase immobilized mixture MWNTs-PDA-AuNPs-AmDH; the mixture was then centrifuged at 8000 rpm for 15 minutes, rinsed, and resuspended in 0.05 M ammonium chloride-ammonia buffer at pH 9.0 to prepare a MWNTs-PDA-AuNPs-AmDH dispersion containing 1 mg / mL amino acid dehydrogenase.

[0065] (5) 10 μL of the MWNTs-PDA-AuNPs-AmDH dispersion obtained in step (4) and the gel containing methylene green, NADH-IL and [BMIM]Cl were dropped on the surface of carbon paper and dried at 4°C to obtain a working electrode, which was recorded as MWNTs-PDA-AuNPs-AmDH-CP. The enzyme loading amount was 10 mg enzyme per 1 g of the composite material.

[0066] (6) Design and performance characterization of bioelectrocatalytic reactor: The amine dehydrogenase electrode MWNTs-PDA-AuNPs-AmDH-CP obtained in step (5) was used as the working electrode, the platinum electrode was used as the counter electrode, and the calomel electrode was used as the reference electrode. Electrochemical experiments were performed at room temperature. The tests of the modified electrode MWNTs-PDA-AuNPs-AmDH-CP were all performed in 10 mL of phosphate buffer (0.02 M, pH 7.0) with the addition of the electron mediator toluidine blue and 20 mg of coenzyme NADH. N2 was introduced before the test. Cyclic voltammetry was used during the test, and no substrate amino acid was added to the blank control. The scan rate of the cyclic voltammetry test was 100 mV / s, the scan range was -1.0 V to 1.0 V, and it was performed in a buffer solution with a pH of 7.0. The substrate α-ketoglutarate was added at a concentration of 200 mM, and the current density was regulated to 1.2 mA / cm 2 After 20 h of reaction, the L-glutamate concentration was 185 mM.

[0067] Example 4

[0068] (1) Preparation of aromatic alcohol dehydrogenase: Based on the original sequence of aromatic alcohol dehydrogenase as shown in SEQ ID NO.3, the gene sequence of aromatic alcohol dehydrogenase was synthesized by a commercial company (Shanghai Bioengineering Co., Ltd.) using PCR method, and used to connect with vector pET28a to construct plasmid. Connection of PCR product and vector pET28a: The PCR product and vector pET28a were double-digested with NdeI and XhoI, and after purification, they were reacted at 4°C overnight under the action of T4 DNA Ligase to obtain a plasmid for transformation. Competent cells were prepared using the Competent Cel Preparation Kit. 10 ng of the transformation plasmid was added to the competent cells, gently mixed, and placed on ice for 30 minutes, placed in a 42°C water bath for 45 seconds, and immediately placed on ice for 1 to 2 minutes. Add LB medium pre-warmed at 37°C, shake and culture at 37°C for 1 hour, take an appropriate amount of bacterial solution to apply to LB solid plates with kanamycin resistance, and culture at 37°C overnight and pick out single colonies, which are recombinant E. coli (E. coli BL21 (DE3) / pET28a).

[0069] Cultivation of recombinant E. coli BL21 (DE3) / pET28a: Inoculate the strain into 200 mL LB medium at a 1% inoculum. The composition of LB medium is 10.0 g / L tryptone, 5.0 g / L yeast powder, 10 g / L NaCl. The culture conditions are: initial pH 7.0, liquid volume fraction is 10%, culture temperature 37 ° C, shaker speed 200 rpm, culture time 6 hours. Add the inducer IPTG to a final concentration of 10 mg / mL, and continue to culture at 30 ° C, 200 rpm for 12 hours.

[0070] Preparation of crude enzyme solution: The fermentation liquid obtained after the cultivation was centrifuged in a refrigerated centrifuge (4°C, 8000rpm, 15min) to obtain cells, discard the supernatant, resuspend the precipitate with Tris-HCl buffer (pH 7.5), wash it thoroughly and centrifuge it, repeat the operation 3 times to obtain cells. The cell liquid was treated with an ultrasonic disruptor, and the probe of the cell disruptor was placed 1cm below the liquid surface. The disruption conditions were ultrasonic for 2 seconds, 4 seconds interval, 40 ultrasonic times, and a power of 200W. Then centrifuge at 4°C and 12,000rpm for 15min to remove insoluble cell debris, and the supernatant was the crude enzyme solution of aromatic alcohol dehydrogenase.

[0071] (2) Preparation of pure aromatic alcohol dehydrogenase: The aromatic alcohol dehydrogenase was separated and purified using a His Trap nickel column from GE, and ultrafiltration and desalting were performed using a 10K ultrafiltration centrifuge tube from PALL. The purification column used in the purification process is a HisTrap HP column that can specifically purify proteins with a His-tagged tag, and the steps include equilibration, loading, equilibration, elution, and column regeneration; the eluted portion was collected and desalted using an ultrafiltration centrifuge tube; the liquid obtained after desalting is the pure aromatic alcohol dehydrogenase (ArylDH with a His-tag) enzyme solution. Tris-HCl (0.05M, pH 7.0) buffer was added thereto to prepare an aqueous solution containing aromatic alcohol dehydrogenase, and the concentration of aromatic alcohol dehydrogenase therein was adjusted to 0.05-50 mg / mL as needed for standby use.

[0072] (3) Synthesis of ionic coenzyme NADH-IL: as in step (3) of Example 1.

[0073] (4) Enzyme immobilization on carbon cloth: TGP-H-060 carbon cloth for fuel cells produced by Toray Industries, Japan, was used as a carrier. Graphene oxide was prepared by the Hummer method, and a uniformly dispersed GO solution was obtained after ultrasonic dispersion for 3 h. 50 mM and 15 mM Zn(NO3)2·3H2O aqueous solutions and 25 mM and 8 mM H3BTC ethanol solutions were prepared respectively. Zn(NO3)2 aqueous solution was added to the GO solution, and the aromatic alcohol dehydrogenase (ArylDH) solution with His-tag obtained in step (2) was added thereto, and finally the ligand H3BTC ethanol solution was added; the carbon paper TGP-H-060 was immersed in the above mixed solution, and CP / Zn3(BTC)2 / GO-ArylDH was obtained by in-situ assembly driven by electrostatic action and metal coordination. 10 μL of gel containing methylene green, NADH-IL and [EMIM]BF4 was dropped on the surface of CP / Zn3(BTC)2 / GO-ArylDH and dried at 4°C to obtain a working modified electrode. Zn3(BTC)2 particles adsorbed on GO sheets have excellent adhesion and are then fixed on carbon paper to prepare three-dimensional electrode materials. The loading amount is 30 mg of enzyme per 1 g of composite material.

[0074] (5) Bioelectrocatalytic reaction: A bioelectrocatalytic microreactor with a capacity of 100 mL was designed based on a disposable plastic test tube. A three-electrode system was designed for the reactor. The carbon paper (3×3 cm) immobilized with enzyme and gel obtained in step (4) was used as the working electrode, the counter electrode was a graphite electrode, and Ag / AgCl was used as the reference electrode. The reaction was carried out in 10 mL of phosphate buffer (0.02 mM, pH 7.4) with NADH-IL added. N2 was introduced before the reaction. Cyclic voltammetry was used during the test, and no substrate was added to the blank control. The scan rate of the cyclic voltammetry test was 50 mV / s, the scan range was -0.8 V to 0.8 V, and it was carried out in a buffer solution with a pH of 7.5. The time of the chronoamperometric current test was 1200 s, and the concentration of acetophenone added was 500 mM. The current density was adjusted to 2.5 mA / cm 2 , react at 40 degrees for 20 hours, and the product phenylethanol is 450mM.

[0075] Example 5

[0076] (1) Preparation of crude malate dehydrogenase: Based on the original sequence of malate dehydrogenase as SEQ ID NO. 4, the gene sequence of malate dehydrogenase was synthesized by a commercial company (Shanghai Bioengineering Co., Ltd.) using PCR method and connected with vector pET28a to construct a plasmid.

[0077] Connection of PCR product (gene sequence of malate dehydrogenase) and vector pET28a: Double digest the PCR product and vector pET28a with NdeI and XhoI, and react overnight at 4°C under the action of T4 DNA ligase after purification to obtain the transformation plasmid. Competent cells were prepared using the Competent Cell Preparation Kit. Add 10 ng of transformation plasmid to the competent cells, mix gently, place on ice for 30 minutes, place in a 42°C water bath for 45 seconds, and immediately place on ice for 1 to 2 minutes. Add LB medium preheated at 37°C, shake and culture at 37°C for 1 hour, take an appropriate amount of bacterial solution to apply to LB solid plates with kanamycin resistance, and pick out single colonies after overnight culture at 37°C, which is the recombinant E. coli (E. coli BL21 (DE3) / pET28a).

[0078] Cultivation of recombinant E. coli BL21 (DE3) / pET28a: Inoculate the strain into 200 mL LB medium at a 1% inoculum. The composition of LB medium is 10.0 g / L tryptone, 5.0 g / L yeast powder, 10 g / L NaCl. The culture conditions are: initial pH 7.0, liquid volume fraction is 10%, culture temperature 37 ° C, shaker speed 200 rpm, culture time 6 hours. Add the inducer IPTG to a final concentration of 10 mg / mL, and continue to culture at 30 ° C, 200 rpm for 2 hours.

[0079] Preparation of crude enzyme solution: The fermentation liquid obtained after the cultivation was centrifuged in a refrigerated centrifuge (4°C, 8000rpm, 15min) to obtain cells, the supernatant was discarded, the precipitate was resuspended with Tris-HCl buffer (pH 7.5), washed thoroughly and centrifuged, and the operation was repeated 3 times to obtain cells. The cell liquid was treated with an ultrasonic disruptor, and the probe of the cell disruptor was placed 1cm below the liquid surface. The disruption conditions were ultrasonic for 2 seconds, 4 seconds interval, 40 ultrasonic times, and a power of 200W. Then, the insoluble cell debris was removed by centrifugation at 4°C and 12,000rpm for 15min, and the supernatant was the crude malate dehydrogenase enzyme solution.

[0080] (2) Preparation of pure malate dehydrogenase enzyme: The crude malate dehydrogenase enzyme solution was separated and purified using GE's His Trap nickel column, and ultrafiltration and desalination were performed using PALL's 10K ultrafiltration centrifuge tube. The purification column used in the purification process is a HisTrap HP column that can specifically purify proteins with His-tagged tags, and the steps include equilibration, loading, equilibration, elution, and column regeneration; the eluted part is collected and desalted using an ultrafiltration centrifuge tube; the liquid obtained after desalting is the pure malate dehydrogenase enzyme liquid. Tris-HCl (0.05M, pH 7.0) buffer is added thereto to prepare a malate dehydrogenase solution containing malate dehydrogenase, and the malate dehydrogenase concentration therein is adjusted to 0.05-50 mg / mL as needed for standby use.

[0081] (3) Preparation of enzyme electrode modification material carbon nanotubes MWNTs-PDA: 30 mg of multi-walled carbon nanotubes (MWNTs) (Nanjing Jicang Nanotechnology Co., Ltd., multi-walled carbon nanotubes JCMT-95-11-10) were added to 30 mL of water and ultrasonically dispersed for 3 h to obtain a uniformly dispersed 1 mg / mL MWNTs solution. 40 mg of PDA was added under stirring and stirred at room temperature for 12 h. After centrifugation and resuspension, a 1 mg / mL MWNTs-PDA solution was prepared. The SEM image of MWNTs-PDA is shown in Figure 2. Figure 6The MWNTs-PDA solution was mixed with the NaAuCl4 solution to obtain the MWNTs-PDA solution containing AuNPs.

[0082] (4) Preparation of MWNTs-PDA-AuNPs-MDH: The malate dehydrogenase solution with an enzyme concentration of 5 mg / mL obtained in step (2) was mixed with the MWNTs-PDA solution containing AuNPs in a ratio of 2:1:1 at 4°C, centrifuged at 8000 rpm and the supernatant discarded to precipitate the malate dehydrogenase immobilized mixture MWNTs-PDA-AuNPs-MDH; the mixture was then centrifuged at 8000 rpm for 15 minutes, rinsed, and resuspended in 0.08 M ammonia-ammonium chloride buffer at pH 9.0 to prepare a MWNTs-PDA-AuNPs-MDH dispersion containing 1 mg / mL malate dehydrogenase.

[0083] (5) Take 10 μL of the MWNTs-PDA-AuNPs-MDH dispersion obtained in step (4) and the gel containing methylene green, NADH-IL and [BMIM]Cl and drop them on the surface of the glassy carbon electrode (GCE), dry them at 4°C, and obtain a modified electrode, which is recorded as MWNTs-PDA-AuNPs-MDH-GCE. As a control, a MWNTs modified electrode was prepared by drop coating method, which is recorded as MWNTs-GCE.

[0084] (6) Bioelectrocatalytic reaction: Based on the glass bioelectrocatalytic microreactor, a three-electrode system of the reactor was designed. The enzyme-fixed GCE obtained in step (5) was used as the working electrode, the counter electrode was a graphite electrode, and Ag / AgCl was used as the reference electrode. The reaction was carried out in 10 mL of phosphate buffer (0.02 mM, pH 7.4) with NADH-IL added. N2 was introduced before the reaction. Cyclic voltammetry was used during the test, and no substrate was added to the blank control. The scan rate of the cyclic voltammetry test was 50 mV / s, the scan range was -0.8 V to 0.8 V, and it was carried out in a buffer solution with a pH of 9.0. The time of the chronoamperometric current test was 1200 s, and the concentration of oxaloacetic acid added was 250 mM. The current density was adjusted to 1.5 mA / cm 2 , react at 10 degrees Celsius for 5 hours, and the concentration of L-malic acid is 220mM.

[0085] Example 6

[0086] (1)~(2)As in Example 5.

[0087] (3) Preparation of immobilized enzyme CP-rGO-PDA-AuNPs-MDH: Graphene oxide was prepared by Hummer method, and a uniformly dispersed GO solution was obtained after ultrasonic dispersion for 3 h. 0.5 g of ascorbic acid was added to 1 mg / mL GO for reduction for 12 h, centrifuged at 12000 rpm, the supernatant was removed, 10 mL of ultrapure water was added for washing, centrifuged again, and 10 mL of 0.1 M citric acid buffer solution (pH 7.0) was added to prepare a 1 mg / mL rGO solution. 20 mg dopamine (DA) and 5 mL 2 mg / mL MDH were added to 10 mL 1 mg / mL rGO solution, and 3.0 mM NaAuCl4 was added under slow stirring at room temperature for chemical oxidation polymerization and immobilization of malate dehydrogenase. After 5 hours of reaction, the obtained dispersion was centrifuged at 10000 rpm, the supernatant was discarded, and the precipitate (i.e., malate dehydrogenase immobilized mixture rGO-PDA-AuNPs-MDH) was dispersed in 10 mL PBS.

[0088] (4) Take 10 μL of the rGO-PDA-AuNPs-MDH dispersion obtained in step (3) and the gel containing methylene green, NADH-IL and [BMIM]Cl and drop them on the surface of carbon paper, dry them at 4°C, and obtain a modified electrode, which is recorded as CP-rGO-PDA-AuNPs-MDH.

[0089] (5) Enzyme electroreactor catalytic reaction: Based on the glass bioelectrocatalytic microreactor, a reactor three-electrode system was designed. The carbon paper CP-rGO-PDA-AuNPs-MDH (2×2 cm) immobilized with enzyme and multifunctional gel obtained in step (4) was used as the working electrode, the counter electrode was a graphite electrode, and Ag / AgCl was used as the reference electrode. The reaction was carried out in 10 mL of phosphate buffer (0.02 mM, pH 7.4) with NADH-IL added. N2 was introduced before the reaction. Cyclic voltammetry was used during the test, and no substrate was added to the blank control. The scan rate of the cyclic voltammetry test was 40 mV / s, the scan range was -0.8 V to 0.8 V, and it was carried out in a buffer solution with a pH of 9.5. The time of the chronoamperometric current test was 1200 s, and the concentration of oxaloacetic acid added was 250 mM. The current density was regulated to 1.5 mA / cm2, the reaction was carried out at 10 degrees Celsius for 5 h, and the concentration of L-malic acid was 120 mM.

[0090] Example 7

[0091] (1) Preparation of crude geraniol dehydrogenase: Based on the original sequence of geraniol dehydrogenase as shown in SEQ ID NO.5, the gene sequence of geraniol dehydrogenase was synthesized by a commercial company (Shanghai Bioengineering Co., Ltd.) using PCR method and connected with vector pET28a to construct a plasmid.

[0092] Connection of PCR product (gene sequence of geraniol dehydrogenase) and vector pET28a: Double digest the PCR product and vector pET28a with NdeI and XhoI, and react overnight at 4°C under the action of T4 DNA ligase after purification to obtain the transformation plasmid. Competent cells were prepared using the Competent Cell Preparation Kit. Add 10 ng of transformation plasmid to the competent cells, mix gently, place on ice for 30 minutes, place in a 42°C water bath for 45 seconds, and immediately place on ice for 1 to 2 minutes. Add LB medium preheated at 37°C, shake and culture at 37°C for 1 hour, take an appropriate amount of bacterial solution to apply to LB solid plates with kanamycin resistance, and pick out single colonies after overnight culture at 37°C, which is the recombinant Escherichia coli (E.coli BL21(DE3) / pET28a).

[0093] Cultivation of recombinant E. coli BL21 (DE3) / pET28a: Inoculate the strain into 200 mL LB medium at a 1% inoculum. The composition of LB medium is 10.0 g / L tryptone, 5.0 g / L yeast powder, 10 g / L NaCl. The culture conditions are: initial pH 7.0, liquid volume fraction is 10%, culture temperature 37 ° C, shaker speed 200 rpm, culture time 6 hours. Add the inducer IPTG to a final concentration of 10 mg / mL, and continue to culture at 30 ° C, 200 rpm for 2 hours.

[0094] Preparation of crude enzyme solution: The fermentation liquid obtained after the cultivation was centrifuged in a refrigerated centrifuge (4°C, 8000rpm, 15min) to obtain cells, the supernatant was discarded, the precipitate was resuspended with Tris-HCl buffer (pH 7.5), washed thoroughly and centrifuged, and the operation was repeated 3 times to obtain cells. The cell liquid was treated with an ultrasonic disruptor, and the probe of the cell disruptor was placed 1cm below the liquid surface. The disruption conditions were ultrasonic for 2 seconds, 4 seconds interval, 40 ultrasonic times, and a power of 200W. Then, the insoluble cell debris was removed by centrifugation at 4°C and 12,000rpm for 15min, and the supernatant was the crude enzyme solution of geraniol dehydrogenase.

[0095] (2) Preparation of pure geraniol dehydrogenase enzyme: The crude geraniol dehydrogenase enzyme solution was separated and purified using a His Trap nickel column from GE, and ultrafiltration and desalting were performed using a 10K ultrafiltration centrifuge tube from PALL. The purification column used in the purification process is a HisTrap HP column that can specifically purify proteins with a His-tagged tag, and the steps include equilibration, loading, equilibration, elution, and column regeneration; the eluted portion was collected and desalted using an ultrafiltration centrifuge tube; the liquid obtained after desalting is the pure geraniol dehydrogenase enzyme liquid. Tris-HCl (0.1M, pH 6.5) buffer was added thereto to prepare a geraniol dehydrogenase solution containing geraniol dehydrogenase, and the geraniol dehydrogenase concentration therein was adjusted to 0.05-50 mg / mL as needed for standby use.

[0096] (3) Enzyme immobilization on carbon cloth: Toray's fuel cell-specific carbon paper TGP-H-060 was used as a carrier. Graphene oxide was prepared by the Hummer method, and a uniformly dispersed GO solution was obtained after ultrasonic dispersion for 3 hours. 50mM and 15mM NiCl2 aqueous solutions and 25mM and 8mM HITP solutions were prepared respectively. NiCl2 aqueous solution was added to the GO solution and the geraniol dehydrogenase (GerDH) solution with His-tag obtained in step (2) was added thereto. The carbon paper TGP-H-060 was immersed in the above mixed solution, and finally the ligand HITP solution was added; CP / Ni3(HITP)2 / GO-GerDH was obtained by electrostatic interaction and metal coordination driven assembly, and a gel containing methylene green, NADH-IL and [EMIM]BF4 was dropped on the surface of CP / Ni3(HITP)2 / GO-GerDH and dried at 4°C to obtain a working modified electrode. Ni3(HITP)2 particles adsorbed on GO sheets have excellent adhesion and are then fixed on carbon paper to prepare three-dimensional electrode materials. The enzyme loading is 40 mg per 1 g of composite material.

[0097] (4) Electrobiocatalytic reaction:

[0098] A bioelectrocatalytic microreactor with a capacity of 100 mL was designed based on a disposable plastic test tube. A three-electrode system of the reactor was designed. The carbon paper (3×3 cm) immobilized with enzyme and gel obtained in step (3) was used as the working electrode, the counter electrode was a graphite electrode, and Ag / AgCl was used as the reference electrode. The reaction was carried out in 10 mL of phosphate buffer (0.02 mM, pH 7.4) with NADH-IL added. N2 was introduced before the reaction. Cyclic voltammetry was used during the test, and no substrate was added to the blank control. The scan rate of the cyclic voltammetry test was 50 mV / s, the scan range was -0.8 V to 0.8 V, and it was carried out in a buffer solution with a pH of 7.0. The time of the chronoamperometric current test was 1200 s, and the concentration of L-phenylpyruvic acid added was 300 mM. The current density was adjusted to 1.5 mA / cm 2 , react at 50°C for 12h, and the concentration of geraniol obtained was 263mM.

[0099] Example 8

[0100] (1) Preparation of crude enzyme solution of homophenylalanine dehydrogenase: According to the homophenylalanine dehydrogenase sequence shown in SEQ ID No. 6, the gene sequence was synthesized by a commercial company (Shanghai Bioengineering Co., Ltd.) using the PCR method; the gene sequence was amplified and connected to the pET28a vector, and then the plasmid was introduced into Escherichia coli E. coli BL21 DE3; Cultivation of recombinant E. coli BL21 (DE3) / pET28a: the strain was inoculated into 200 mL LB medium at a 1% inoculation rate. The composition of LB medium is 10.0 g / L tryptone, 5.0 g / L yeast extract, and 10 g / L NaCl. The culture conditions are: initial pH 7.0, liquid volume fraction of 10%, culture temperature 37°C, shaker speed 200 rpm, and culture time 6 hours. The inducer IPTG was added to a final concentration of 10 mg / mL, and the culture was continued at 25°C and 200 rpm for 12 hours. The fermentation liquid obtained after the cultivation was centrifuged in a refrigerated centrifuge (4°C, 8000rpm, 15min) to obtain cells, the supernatant was discarded, the precipitate was resuspended with phosphate buffer (pH=7-7.4), washed thoroughly and centrifuged, the operation was repeated 3 times, and a cell suspension with a concentration of 50-150g / L was prepared with phosphate buffer (pH 7-7.4). The prepared cell suspension was placed in an ice bath, and the cell liquid was treated with an ultrasonic disruptor. The probe of the cell disruptor was placed 1cm below the liquid surface. The disrupting conditions were ultrasonic for 3 seconds, 6 seconds interval, 60 ultrasonic times, and a power of 200W. Then, the insoluble cell debris was removed by centrifugation at 4°C and 12,000rpm for 15min, and the supernatant was the crude enzyme solution containing high phenylalanine dehydrogenase with a His-tag label.

[0101] (2) Preparation of pure homophenylalanine dehydrogenase: GE His Trap nickel column (Histrap TM HP, 5 mL) was used to separate and purify the crude enzyme solution obtained in step (1), and ultrafiltration and desalting were performed using a 10K ultrafiltration centrifuge tube from PALL. The purification process used a purification column that can specifically purify proteins with His-tagged tags, and the steps included balancing, loading, balancing, eluting, and column regeneration; the eluted portion was collected and desalted using an ultrafiltration centrifuge tube; the liquid obtained after desalting was the purified Figure 2 Pure enzyme solution of His-tag homophenylalanine dehydrogenase is shown.

[0102] (3) Enzyme activity detection: The catalytic reaction system contains 40mM NADH, 0.2mol / L glycine-sodium hydroxide buffer solution (pH 9.5), 20% isopropanol as the cosolvent, 40mM ethyl 2-oxo-4-phenylbutyrate and 20mg / mL enzyme, and the reaction is carried out at 37°C. The enzyme activity is measured at a wavelength of 340nm. Enzyme activity is defined as the oxidation consumption (or generation) of 1μmol NAD per minute under the above conditions. + The amount of enzyme required is one unit of enzyme activity. The measured enzyme activity is 0.192U / mg.

[0103] (4) Enzyme activity detection: The catalytic reaction system contains 200mM NADH, 0.5mol / L glycine-sodium hydroxide buffer solution (pH 11), 10% acetonitrile as the cosolvent, 80mM L-2-carbonyl-5-phenylvaleric acid and 60mg / mL enzyme, and the reaction is carried out at 40°C. The enzyme activity is measured at a wavelength of 340nm. Enzyme activity is defined as the oxidation consumption (or generation) of 1μmol NAD per minute under the above conditions. + The amount of enzyme required is one unit of enzyme activity. The enzyme activity was determined to be 0.223U / mg.

[0104] (5) In situ mineralization and enzyme immobilization on porous composite carbon materials

[0105] The enzyme and gel were immobilized on porous carbon paper, and the electrode / electron mediator / coenzyme / enzyme integrated composite system was constructed by biomimetic mineralization. First, a 100 mg / mL 500 kDa branched polyethyleneimine solution was prepared to prepare graphene oxide-polyethyleneimine (GO-PEI) composite material, and rGO-PEI was obtained by hydrothermal reduction. Secondly, high phenylalanine dehydrogenase was added with Mn 2+ The metal salt solution was made into a final concentration of 50mmol / L, and was immobilized on the rGO-PEI carrier by MnCl2 metal coordination. 2mg / mL high phenylalanine dehydrogenase solution was added to rGO-PEI, and PEI and Mn 2+Ionic coordination force to immobilize enzymes. A titanium precursor di-(2-hydroxypropionic acid) diammonium dihydroxide solution (Ti-BALDH) with a concentration of 0.5 mol / L and a pH range of 10.0 and a multifunctional conductive gel containing methylene green, NADH-IL and [EMIM]BF4 were added, and PEI induced TiO2 biomimetic mineralization to achieve in situ immobilization of enzymes and coenzymes on porous carbon paper.

[0106] (6) Electrocatalytic reaction: Enzyme activity detection: The catalytic reaction system contains 100 mM NADH, 0.6 mol / L glycine-sodium hydroxide buffer solution (pH 11), 30% propanol as a cosolvent, 40 mM L-2-carbonyl-3-phenylbutyric acid and 80 mg / ml enzyme, and the reaction is carried out at 35°C. The enzyme activity is measured at a wavelength of 340 nm. Enzyme activity is defined as the oxidation consumption (or generation) of 1 μmol NAD per minute under the above conditions. + The amount of enzyme required is one enzyme activity unit. The enzyme activity was determined to be 0.22U / mg. A bioelectrocatalytic microreactor with a capacity of 50mL was designed based on disposable plastic test tubes. A three-electrode system for the reactor was designed, with carbon paper (5×5cm) fixed with enzymes and gel as the working electrode, a graphite electrode as the counter electrode, and Ag / AgCl as the reference electrode. The reaction was carried out in 10mL phosphate buffer (0.02mM, pH 7.4) with NADH-IL added. N2 was introduced before the reaction. Cyclic voltammetry was used during the test, in which no substrate was added to the blank control. The scan rate of the cyclic voltammetry test was 50mV / s, the scan range was -0.8V to 0.8V, and it was carried out in a buffer solution with a pH of 9.5. The time of the chronoamperometric current test was 1200s, and L-2-carbonyl-3-phenylbutyric acid was added at a concentration of 40mM. The current density was regulated to 1.5mA / cm 2 , 20 degrees for 10 hours, to obtain 33.7mM L-2-amino-3-phenylbutyric acid. The product was analyzed by NMR, and the NMR spectrum was obtained. 1H NMR: δ1.26 (3H, d, J = 6.7Hz), 3.18 (1H, dq, J = 7.1, 6.7Hz), 3.64 (1H, d, J = 7.1Hz), 7.21 (2H, dddd, J = 7.8, 1.3, 1.2, 0.5Hz), 7.28-7.40 (3H, 7.35 (dddd, J = 7.8, 7.7, 1.9, 0.5Hz), 7.31 (tt, J = 7.7, 1.3Hz)). It is consistent with the characteristics of the product L-2-amino-3-phenylbutyric acid.

[0107] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention. Sequence Listing <110> Xiamen University <120> Redox enzyme electrode for enzyme electrocatalytic reduction and preparation method thereof and enzyme electroreactor thereof <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 354 <212> PRT <213> Artificial Sequence <400> 1 Met Lys Ile Leu Glu Thr Met Lys Ala Ser Asp Tyr Glu Gln Leu Val 1 5 10 15 Phe Cys Gln Asp Glu Lys Thr Gly Leu Lys Gly Ile Ile Ala Ile His 20 25 30 Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr 35 40 45 Asp Asn Glu Glu Glu Ala Ile Glu Asp Val Leu Arg Leu Ala Arg Gly 50 55 60 Met Thr Tyr Lys Ser Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Gly Val Ile Ile Gly Asp Pro Lys Lys Asp Lys Ser Glu Glu Met Trp 85 90 95 Arg Ala Phe Gly Arg Phe Val Gln Ser Leu Asn Gly Arg Tyr Ile Thr 100 105 110 Ala Glu Asp Val Gly Val Arg Glu Thr Asp Leu Glu Ile Val Asn Thr 115 120 125 Glu Thr Asp Phe Ala Val Gly Leu Pro Gly Lys Ser Gly Asn Pro Ser 130 135 140 Pro Ala Thr Ala Tyr Gly Val Tyr Ser Gly Ile Lys Ala Val Ala Asp 145 150 155 160 Glu Ile Trp Gly Ser Ala Asp Leu Asn Gly Lys Thr Ile Ala Ile Gln 165 170 175 Gly Ala Gly Ser Val Gly Tyr Tyr Leu Ser Glu Leu Leu His Lys Asp 180 185 190 Gly Ala Lys Leu Ile Val Thr Asp Ile Asp Lys Glu Ala Val Asp Lys 195 200 205 Leu Val Ser Asp Phe Gly Ala Thr Ala Val Glu Thr Asp Glu Ile Tyr 210 215 220 Glu Gln Glu Ala Asp Ile Phe Ala Pro Cys Ala Leu Gly Ala Ile Leu 225 230 235 240 Asn Asp Glu Thr Ile Pro Lys Leu Lys Val Lys Ala Val Ala Gly Ala 245 250 255 Ala Asn Asn Gln Leu Glu Asp Glu Lys Arg His Ala Glu Glu Leu Lys 260 265 270 Lys Arg Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala Gly Gly 275 280 285 Val Ile Asn Val Ser Phe Glu Leu Thr Gly Tyr Asp Glu Glu Arg Ala 290 295 300 Tyr Arg Lys Ile Ser Thr Ile Tyr Asp Asn Ile Lys Lys Ile Phe Asn 305 310 315 320 Ile Ala Asn Arg Asp Asp Ile Thr Ser His Glu Ala Ala Asn Arg Met 325 330 335 Ala Glu Glu Arg Ile Glu Ala Ile Lys His Val Lys Thr Ser Tyr Ile 340 345 350 Asn Lys <210> 2 <211> 1152 <212> DNA <213> Artificial Sequence <400> 2 catatgagct tagtagaaaa aacatccatc ataaaagatt tcactctttt tgaaaaaatg 60 tctgaacatg aacaagttgt tttttgcaac gatccggcga caggactaag ggccattatc 120 gctattcatg acaccacact cggacctgcg ctcggcggct gccgcatgca gccttataac 180 agtgtggaag aagcattgga agatgctctt cgcctttcca aaggaatgac ttactcttgc 240 gcggcgtccg atgtcgactt tggcggcgga aaagcagtca ttatcggtga tccgcagaaa 300 gataaatctc cagaactgtt ccgcgcgttt ggccaatttg ttgattcgct tggcggccgt 360 ttctatacag gtactgatat gggaacgaat atggaagatt tcattcacgc catgaaagaa 420 acaaactgca ttgttggggt gccggaagct tacggcggcg gcggagattc ctctattcca 480 actgccatgg gtgtcctgta cggcattaaa gcaaccaaca aaatgttgtt tggcaaggac 540 gatcttggcg gcgtcactta tgccattcaa ggacttggca aagtaggcta caaagtagcg 600 gaagggctgc tcgaagaagg tgctcattta tttgtaacgg atattaacga gcaaacgttg 660 gaggctatcc aggaaaaagc aaaaacaaca tccggttctg tcacggtagt agcgagcgat 720 gaaatttatt cccaggaagc cgatgtgttc gttccgtgtg catttggcgg cgttgttaat 780 gatgaaacga tgaagcagtt caaggtgaaa gcaatcgccg gttcagccct gaatcagctg 840 cttacggagg atcacggcag acaccttgca gacaaaggca ttctgtatgc tccggattat 900 attgttaact ctggcggtct gatccaagta gccgacgaat tgtatgaggt gaacaaagaa 960 cgcgtgcttg cgaagacgaa gcatatttac gacgcaattc ttgaagtgta ccagcaagcg 1020 gaattagatc aaatcaccac aatggaagca gccaacagaa tgtgtgagca aagaatggcg 1080 gcaagaggcc gacgcaacag cttctttact tcttctgtta agccaaaatg ggatattcgc 1140 aactaactcg ag 1152 <210> 3 <211> 366 <212> PRT <213> Artificial Sequence <400> 3 Met Glu Ile Lys Ala Ala Ile Val Arg Gln Lys Asn Gly Pro Phe Leu 1 5 10 15 Leu Glu His Val Ala Leu Asn Glu Pro Ala Glu Asp Gln Val Leu Val 20 25 30 Arg Leu Val Ala Thr Gly Leu Cys His Thr Asp Leu Val Cys Arg Asp 35 40 45 Gln His Tyr Pro Val Pro Leu Pro Met Val Phe Gly His Glu Gly Ala 50 55 60 Gly Val Val Glu Arg Val Gly Ser Ala Val Lys Lys Val Gln Pro Gly 65 70 75 80 Asp His Val Val Leu Thr Phe Tyr Thr Cys Gly Ser Cys Asp Ala Cys 85 90 95 Leu Ser Gly Asp Pro Thr Ser Cys Ala Asn Ser Phe Gly Pro Asn Phe 100 105 110 Met Gly Arg Ser Val Thr Gly Glu Cys Thr Ile His Asp His Gln Gly 115 120 125 Ala Glu Val Gly Ala Ser Phe Phe Gly Gln Ser Ser Phe Ala Thr Tyr 130 135 140 Ala Leu Ser Tyr Glu Arg Asn Thr Val Lys Val Thr Lys Asp Val Pro 145 150 155 160 Leu Glu Leu Leu Gly Pro Leu Gly Cys Gly Ile Gln Thr Gly Ala Gly 165 170 175 Ser Val Leu Asn Ala Leu Asn Pro Pro Ala Gly Ser Ala Ile Ala Ile 180 185 190 Phe Gly Ala Gly Ala Val Gly Leu Ser Ala Val Met Ala Ala Val Val 195 200 205 Ala Gly Cys Thr Thr Ile Ile Ala Val Asp Val Lys Glu Asn Arg Leu 210 215 220 Glu Leu Ala Ser Glu Leu Gly Ala Thr His Ile Ile Asn Pro Ala Ala 225 230 235 240 Asn Asp Pro Ile Glu Ala Ile Lys Glu Ile Phe Ala Asp Gly Val Pro 245 250 255 Tyr Val Leu Glu Thr Ser Gly Leu Pro Ala Val Leu Thr Gln Ala Ile 260 265 270 Leu Ser Ser Ala Ile Gly Gly Glu Ile Gly Ile Val Gly Ala Pro Pro 275 280 285 Met Gly Ala Thr Val Pro Val Asp Ile Asn Phe Leu Leu Phe Asn Arg 290 295 300 Lys Leu Arg Gly Ile Val Glu Gly Gln Ser Ile Ser Asp Ile Phe Ile 305 310 315 320 Pro Arg Leu Val Glu Leu Tyr Arg Gln Gly Lys Phe Pro Phe Asp Lys 325 330 335 Leu Ile Lys Phe Tyr Pro Phe Asp Glu Ile Asn Arg Ala Ala Glu Asp 340 345 350 Ser Glu Lys Gly Val Thr Leu Lys Pro Val Leu Arg Ile Gly 355 360 365 <210> 4 <211> 305 <212> PRT <213> Artificial Sequence <400> 4 Thr Lys Val Ser Val Val Gly Ala Ala Gly Thr Val Gly Ala Ala Ala 1 5 10 15 Gly Tyr Asn Ile Ala Leu Arg Asp Ile Ala Asp Glu Val Val Phe Val 20 25 30 Asp Ile Pro Asp Lys Glu Asp Asp Thr Val Gly Gln Ala Ala Asp Thr 35 40 45 Asn His Gly Ile Ala Tyr Asp Ser Asn Thr Arg Val Arg Gln Gly Gly 50 55 60 Tyr Glu Asp Thr Ala Gly Ser Asp Val Val Val Ile Thr Ala Gly Ile 65 70 75 80 Pro Arg Gln Pro Gly Gln Thr Arg Ile Asp Leu Ala Gly Asp Asn Ala 85 90 95 Pro Ile Met Glu Asp Ile Gln Ser Ser Leu Asp Glu His Asn Asp Asp 100 105 110 Tyr Ile Ser Leu Thr Thr Ser Asn Pro Val Asp Leu Leu Asn Arg His 115 120 125 Leu Tyr Glu Ala Gly Asp Arg Ser Arg Glu Gln Val Ile Gly Phe Gly 130 135 140 Gly Arg Leu Asp His Asn Arg Ala Lys Ala Gln Leu Ala Lys Lys Thr 145 150 155 160 Gly Thr Gly Val Asp Arg Ile Arg Arg Met Thr Val Ile Leu Gly Glu 165 170 175 His Gly Asp Ala Gln Val Pro Val Phe Ser Lys Val Ser Val Asp Gly 180 185 190 Thr Asp Pro Glu Phe Ser Gly Asp Glu Lys Glu Gln Leu Leu Gly Asp 195 200 205 Leu Gln Glu Ser Ala Met Asp Val Ile Glu Arg Lys Gly Ala Thr Glu 210 215 220 Trp Gly Pro Ala Arg Gly Val Ala His Met Val Glu Ala Ile Leu His 225 230 235 240 Asp Thr Gly Glu Val Leu Pro Ala Ser Val Lys Leu Glu Gly Glu Phe 245 250 255 Gly His Glu Asp Thr Ala Phe Gly Val Pro Val Ser Leu Gly Ser Asn 260 265 270 Gly Val Glu Glu Ile Val Glu Trp Asp Leu Asp Asp Tyr Glu Gln Asp 275 280 285 Leu Met Ala Asp Ala Ala Glu Lys Leu Ser Asp Gln Tyr Asp Lys Ile 290 295 300 Ser 305 <210> 5 <211> 373 <212> PRT <213> Artificial Sequence <400> 5 Met Asn Asp Thr Gln Asp Phe Ile Ser Ala Gln Ala Ala Val Leu Arg 1 5 10 15 Gln Val Gly Gly Pro Leu Ala Val Glu Pro Val Arg Ile Ser Met Pro 20 25 30 Lys Gly Asp Glu Val Leu Ile Arg Ile Ala Gly Val Gly Val Cys His 35 40 45 Thr Asp Leu Val Cys Arg Asp Gly Phe Pro Val Pro Leu Pro Ile Val 50 55 60 Leu Gly His Glu Gly Ser Gly Thr Val Glu Ala Val Gly Glu Gln Val 65 70 75 80 Arg Thr Leu Lys Pro Gly Asp Arg Val Val Leu Ser Phe Asn Ser Cys 85 90 95 Gly His Cys Gly Asn Cys His Asp Gly His Pro Ser Asn Cys Leu Gln 100 105 110 Met Leu Pro Leu Asn Phe Gly Gly Ala Gln Arg Val Asp Gly Gly Gln 115 120 125 Val Leu Asp Gly Ala Gly His Pro Val Gln Ser Met Phe Phe Gly Gln 130 135 140 Ser Ser Phe Gly Thr His Ala Val Ala Arg Glu Ile Asn Ala Val Lys 145 150 155 160 Val Gly Asp Asp Leu Pro Leu Glu Leu Leu Gly Pro Leu Gly Cys Gly 165 170 175 Ile Gln Thr Gly Ala Gly Ala Ala Ile Asn Ser Leu Gly Ile Gly Pro 180 185 190 Gly Gln Ser Leu Ala Ile Phe Gly Gly Gly Gly Val Gly Leu Ser Ala 195 200 205 Leu Leu Gly Ala Arg Ala Val Gly Ala Asp Arg Val Val Val Ile Glu 210 215 220 Pro Asn Ala Ala Arg Arg Ala Leu Ala Leu Glu Leu Gly Ala Ser His 225 230 235 240 Ala Leu Asp Pro His Ala Glu Gly Asp Leu Val Ala Ala Ile Lys Ala 245 250 255 Ala Thr Gly Gly Gly Ala Thr His Ser Leu Asp Thr Thr Gly Leu Pro 260 265 270 Pro Val Ile Gly Ser Ala Ile Ala Cys Thr Leu Pro Gly Gly Thr Val 275 280 285 Gly Met Val Gly Leu Pro Ala Pro Asp Ala Pro Val Pro Ala Thr Leu 290 295 300 Leu Asp Leu Leu Ser Lys Ser Val Thr Leu Arg Pro Ile Thr Glu Gly 305 310 315 320 Asp Ala Asp Pro Gln Arg Phe Ile Pro Arg Met Leu Asp Phe His Arg 325 330 335 Ala Gly Lys Phe Pro Phe Asp Arg Leu Ile Thr Arg Tyr Arg Phe Asp 340 345 350 Gln Ile Asn Glu Ala Leu His Ala Thr Glu Lys Gly Glu Ala Ile Lys 355 360 365 Pro Val Leu Val Phe 370 <210> 6 <211> 354 <212> PRT <213> Artificial Sequence <400> 6 Met Phe Glu Lys Ile Ser Gln His Glu Gln Val Val Phe Cys Asn Asp 1 5 10 15 Pro Ser Thr Gly Leu Lys Ala Ile Ile Ala Ile His Asn Thr Thr Leu 20 25 30 Gly Pro Ala Leu Gly Gly Cys Arg Met Arg Pro Tyr Gly Ser Val Asp 35 40 45 Glu Ala Leu Glu Asp Val Leu Arg Leu Ser Lys Gly Met Thr Tyr Lys 50 55 60 Cys Ala Gly Ala Asp Val Asp Phe Gly Gly Gly Lys Ser Val Ile Ile 65 70 75 80 Gly Asp Pro Met Thr Asp Arg Thr Pro Glu Leu Phe Arg Ala Phe Gly 85 90 95 Gln Phe Val Asp Ser Leu Asn Gly Arg Phe Tyr Thr Gly Thr Asp Met 100 105 110 Gly Thr Thr Pro Asp Asp Phe Met His Ala Leu Lys Glu Thr Asn Cys 115 120 125 Ile Val Gly Val Pro Glu Glu Tyr Gly Gly Ser Gly Asp Ser Ser Val 130 135 140 Pro Thr Ala Gln Gly Val Ile Tyr Gly Leu Gln Ala Thr Ile Gln Thr 145 150 155 160 Leu Glu Gly Thr Asp Glu Leu Ser Gly Lys Ser Tyr Ser Ile Gln Gly 165 170 175 Leu Gly Lys Val Gly Phe Lys Val Ala Glu Gln Leu Leu Ala Ala Gly 180 185 190 Ala Gln Ile Tyr Val Thr Asp Ile Asn Glu Lys Ala Leu Lys Met Ile 195 200 205 Gln Glu Arg Ala Glu Leu Leu Pro Gly Asn Val Glu Val Val Glu Gly 210 215 220 Ser Asp Ile Tyr Gly Val Asp Ala Asp Ile Phe Ile Pro Cys Ala Leu 225 230 235 240 Gly Gly Ile Ile His Asp Glu Thr Ile Glu Gln Leu Lys Val Lys Ala 245 250 255 Ile Val Gly Ser Ala Asn Asn Gln Leu Leu Glu Asp Lys His Gly Leu 260 265 270 Tyr Leu Gln Gln Lys Gly Ile Leu Tyr Gly Pro Asp Tyr Ile Val Asn 275 280 285 Ala Gly Gly Leu Ile Gln Val Ala Asp Glu Leu Tyr Gly Pro Asn Lys 290 295 300 Ala Arg Val Leu Thr Lys Thr Arg Ala Ile Tyr Asp Ser Leu Ile Gln 305 310 315 320 Ile Tyr Ser Glu Ser Thr Lys Asn Gln Ile Ser Thr Met Glu Ala Ala 325 330 335 Asn Leu Phe Cys Glu Glu Lys Leu Leu Ala Arg Ser Lys Arg Asn Ser 340 345 350 Phe Phe

Claims

1. A redox enzyme electrode for enzyme electrocatalytic reduction, characterized in that: It comprises a substrate electrode, an electron conductor, an electron mediator, an enzyme and a coenzyme; the electron conductor comprises a combination of reduced graphene oxide, polyethyleneimine and a biomimetic mineralized TiO2 material; the enzyme is homophenylalanine dehydrogenase, and the coenzyme is NADH-IL; the coenzyme and the enzyme are fixed together on the enzyme electrode; The preparation method of the redox enzyme electrode comprises: compounding an immobilized mixture of enzyme and electron conductor with electron mediator and coenzyme on the surface of the substrate electrode to obtain the redox enzyme electrode; coordinating and fixing the enzyme on reduced graphene oxide-polyethyleneimine, then adding Ti-BALDH, inducing TiO2 bionic mineralization through polyethyleneimine to immobilize the enzyme, and obtaining an immobilized mixture of enzyme and electron conductor; wherein, Firstly, 100 mg / mL 500 kDa branched polyethyleneimine solution was prepared to prepare graphene oxide-polyethyleneimine composite material, and rGO-PEI was obtained by hydrothermal reduction. Secondly, enzyme was added to Mn 2+ The metal salt solution was made into a final concentration of 50mmol / L, and was immobilized on the rGO-PEI carrier by MnCl2 metal coordination. 2mg / mL enzyme solution was added to rGO-PEI, and PEI and Mn 2+ The enzyme is immobilized by ion coordination force; a titanium precursor di-(2-hydroxypropionic acid) diammonium dihydroxide solution with a concentration of 0.5 mol / L and a pH range of 10.0 and a multifunctional conductive gel containing methylene green, NADH-IL and [EMIM]BF4 are added, and PEI induces TiO2 biomimetic mineralization to achieve in situ immobilization of enzymes and coenzymes on the substrate.

2. The redox enzyme electrode according to claim 1, characterized in that: The base electrode is a carbon paper electrode.

3. A method for preparing an oxidoreductase electrode according to any one of claims 1 or 2, characterized in that: The immobilized mixture of enzyme and electron conductor is compounded with electron mediator and coenzyme on the surface of the substrate electrode to obtain the redox enzyme electrode.

4. The preparation method according to claim 3, characterized in that: The preparation method of the immobilized mixture of enzyme and electron conductor comprises: fixing the enzyme on reduced graphene oxide-polyethyleneimine by coordination, then adding Ti-BALDH, inducing TiO2 bionic mineralization by polyethyleneimine to immobilize the enzyme, and obtaining the immobilized mixture of enzyme and electron conductor.

5. The preparation method according to claim 3, characterized in that: The NADH-IL forms a gel with the electron mediator, and the immobilized mixture of the enzyme and the electron conductor is complexed with the gel on the surface of the substrate electrode.

6. An enzyme electroreactor for enzyme electrocatalytic reduction, characterized in that: The enzyme-electroreactor adopts a three-electrode system, with the redox enzyme electrode according to any one of claims 1 or 2 as the working electrode; the reference electrode is selected from a saturated calomel electrode, a hydrogen electrode, a silver|silver chloride electrode or a mercury|mercuric oxide electrode; and the counter electrode is a platinum wire electrode or a carbon electrode.

7. A method for catalytic reduction reaction using the enzyme electroreactor according to claim 6, characterized in that: The reaction is carried out in a cyclic voltammetry and / or chronoamperometry manner; the potential scanning rate of the cyclic voltammetry is 1 to 500 mV / s; the reaction is carried out in a buffer added with 1 to 20 mg of coenzyme NADH, N2 is introduced before the reaction, the reaction substrate is keto acid, a substrate of amino acid dehydrogenase; the concentration range of the substrate is 5 mM to 500 mM; the buffer is at least one of phosphate buffer, borate buffer, citrate buffer, carbonate buffer, and Tris-HCl buffer.

8. The method according to claim 7, characterized in that: A 1.5-100 mL three-electrode system was used, with carbon paper immobilized with enzyme and gel as the working electrode, a platinum wire electrode as the counter electrode, and Ag / AgCl as the reference electrode. The reaction was carried out in 0.5-100 mL phosphate buffer added with NADH-IL, and N2 was introduced before the reaction. Cyclic voltammetry was used during the test, and no substrate was added as a blank control. The scan rate of the cyclic voltammetry test was 40-60 mV / s, the scan range was -1 V to 1 V, and the test was carried out in a buffer solution with a pH of 6.8 to 7.

2. The time of the chronoamperometric current test was 30-1800 s, and the substrate was added every 25-35 s. Adjustable current density 0.1~1.5 mA / cm 2 The total conversion number of coenzyme regeneration was calculated and the electrochemical regeneration conditions were optimized; the voltage was 1-200 mV / s, the scanning range was -1 V to 1 V, and the pH was 6.0-12.0 in a buffer solution. The time of the chronoamperometric current test was 30-1800 s, 1140-1260 s, and the substrate was added once at 1 mM to 500 mM; the current density was adjusted to 0.1-1.5 mA / cm 2 .

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