Porous composite enzyme fixed electrode and preparation method and application thereof, continuous flow enzyme electrocatalytic reaction system and application thereof

Abstract

The present invention belongs to the field of enzyme bioelectrocatalysis technology, and specifically relates to a porous composite enzyme fixed electrode, a preparation method and application thereof, a continuous flow enzyme electrocatalytic reaction system and application thereof. The porous composite enzyme fixed electrode provided by the present invention comprises a foamed metal, a porous conductive coating arranged on the surface of the foamed metal; and a composite enzyme loaded in the pore structure of the porous conductive coating. The present invention realizes efficient loading and effective fixation of multiple enzymes of coenzyme reductase, coenzyme and oxidoreductase on the fixed electrode through the characteristics of the micron-nano multi-level pore structure, and the composite enzyme has higher stability on the carrier under the condition of continuous flow, and at the same time realizes effective fixation of the composite enzyme and increases the effective contact area between the composite enzyme and the continuously flowing reaction solution, thereby, the porous composite enzyme fixed electrode provided by the present invention can have efficient enzyme reaction efficiency under continuous flow conditions.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme bioelectrocatalysis, and specifically relates to a porous composite enzyme fixed electrode and a preparation method and application thereof, and a continuous flow enzyme electrocatalytic reaction system and application thereof. Background Art

[0002] As a green and efficient catalytic technology, enzyme biophotoelectrocatalysis combines the advantages of bioenzyme catalysis and photoelectrocatalysis. Compared with traditional enzyme catalysis, enzyme biophotoelectrocatalysis is more flexible and convenient because it couples electrode reactions with redox enzyme catalysis and does not require the addition of a large amount of coenzyme factors. Enzyme bioelectrocatalysis uses redox enzymes as biocatalysts to catalyze redox reactions on electrodes, thereby achieving the conversion of chemical energy and electrical energy. Enzyme bioelectrocatalysis has the advantages of high activity, selectivity and mild reaction conditions of biocatalysts, as well as the ability to use renewable electricity and light energy as electron sources and achieve high energy conversion efficiency through electrocatalysis. This technology has been widely studied for applications such as fine chemical synthesis, CO2 fixation, and sensing and monitoring, opening up new research directions in chemical synthesis, biofuels and materials.

[0003] At present, the enzyme biophotoelectrocatalytic reactor used in laboratory research is an H-type double-chamber reactor. However, it is difficult to expand to pilot or industrial scale due to its slow diffusion at the solid-liquid interface, low reaction efficiency of active sites on the electrode surface, and high internal resistance.

[0004] The continuous flow electrolytic cell based on membrane electrode assembly has a higher flux, can achieve continuous production, and is more suitable for large-scale industrial applications. However, when enzyme biophotocatalysis is applied to the continuous flow reaction system, there are still problems such as poor stability of enzyme fixed electrode (serious enzyme shedding) and low efficiency caused by small reaction specific surface area, which leads to the inability of continuous flow enzyme electrocatalytic reaction system based on membrane electrode assembly to operate efficiently and stably. Summary of the invention

[0005] The purpose of the present invention is to provide a porous composite enzyme fixed electrode and its preparation method and application, a continuous flow enzyme electrocatalytic reaction system and application. The porous composite enzyme fixed electrode provided by the present invention can realize the effective and stable fixation of various types of enzymes under continuous flow conditions, and has high enzyme reaction efficiency, and can also realize the cofactor regeneration function, thereby effectively reducing the synthesis cost of the continuous flow enzyme electrocatalytic reaction.

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

[0007] The present invention provides a porous composite enzyme fixed electrode, comprising a foamed metal, a porous conductive coating arranged on the surface of the foamed metal; and a composite enzyme loaded in the pore structure of the porous conductive coating; the pore structure of the foamed metal is a micrometer-level pore structure, the pore structure of the porous conductive coating is a nanometer-level pore structure, and the porous conductive coating is formed by nano-conductive fillers; the composite enzyme comprises coenzyme reductase, coenzyme and oxidoreductase.

[0008] Preferably, the average pore size of the foamed metal is 20-50 μm, and the porosity is 40-90%.

[0009] Preferably, the thickness of the porous conductive coating is 1 to 3 μm; the average pore size of the porous conductive coating is 20 to 80 nm, and the porosity is 20 to 50%.

[0010] Preferably, the nano-conductive filler includes one or more of nano-silver, nano-gold, nano-indium tin oxide, graphene and carbon nanotubes.

[0011] Preferably, the coenzyme includes one or more of adenosine triphosphate, reduced coenzyme II and reduced coenzyme I.

[0012] The present invention provides a method for preparing the porous composite enzyme-immobilized electrode described in the above technical solution, comprising the following steps:

[0013] (1) impregnating the foamed metal into a conductive filler slurry, pre-calcining the obtained wet blank in a protective atmosphere and then standing to solidify to obtain an initial solidified embryo; the conductive filler slurry comprises a nano conductive filler, an organic solvent, a binder and a dispersant;

[0014] (2) Repeating the steps of impregnation, pre-calcination and standing curing in step (1) on the initially cured embryo until a conductive cured coating is formed on the surface of the foamed metal;

[0015] (3) calcining the foamed metal having a conductive cured coating on the surface in a protective atmosphere to form a porous conductive coating on the surface of the foamed metal;

[0016] (4) Immersing a foamed metal having a porous conductive coating on its surface into a complex enzyme solution to obtain the porous complex enzyme fixed electrode; the complex enzyme solution comprises coenzyme reductase, coenzyme, oxidoreductase and buffer solution.

[0017] Preferably, the binder includes one or more of epoxy resin, methyl hexahydrophthalic anhydride and polyetherimide;

[0018] The dispersant includes polyvinyl pyrrolidone and / or polyvinyl alcohol;

[0019] The organic solvent includes one or more of acetone, terpinene and N,N-dimethylformamide.

[0020] Preferably, the pre-calcination temperature is 250-400°C; the calcination temperature is 600-800°C.

[0021] The present invention provides a continuous flow enzyme electrocatalytic reaction system, comprising a cathode fixed shell 2, a first waterproof membrane 10, a cathode electrode sheet 3, a second waterproof membrane 12, a cathode reaction chamber 13, a third waterproof membrane 14, an ion exchange membrane 5, a fourth waterproof membrane 15, an anode reaction chamber 16, a fifth waterproof membrane 6, an anode electrode sheet 17, a sixth waterproof membrane 18 and an anode fixed shell 7, which are fixedly connected in sequence; the cathode reaction chamber 13 or the anode reaction chamber 16 is provided with the porous composite enzyme fixed electrode described in the above technical scheme or the porous composite enzyme fixed electrode prepared by the preparation method described in the above technical scheme.

[0022] The present invention provides the use of the porous composite enzyme fixed electrode described in the above technical scheme or the porous composite enzyme fixed electrode prepared by the preparation method described in the above technical scheme or the continuous flow enzyme electrocatalytic reaction system described in the above technical scheme in enzyme electrocatalytic synthesis of chemical substances.

[0023] The present invention provides a porous composite enzyme fixed electrode, comprising a foamed metal, a porous conductive coating arranged on the surface of the foamed metal; and a composite enzyme loaded in the pore structure of the porous conductive coating; the pore structure of the foamed metal is a micrometer-level pore structure, the pore structure of the porous conductive coating is a nanometer-level pore structure, and the porous conductive coating is formed by nano-conductive fillers; the composite enzyme comprises coenzyme reductase, coenzyme and oxidoreductase. The present invention uses a foamed metal with a micron-sized pore structure as a support for a composite enzyme electrode, and sets a conductive coating with a nano-sized pore structure on the surface of the micron-sized porous structure support, and finally loads the composite enzyme through the nano-sized pore structure of the conductive coating; the present invention realizes the efficient loading and effective fixation of multiple enzymes of coenzyme reductase, coenzyme and oxidoreductase on the fixed electrode through the characteristics of the micron-nano multi-level pore structure, and the stability of the composite enzyme on the carrier is higher under the condition of continuous flow, thereby improving the reaction activity of the fixed enzyme electrode; at the same time, the characteristics of the micron-nano multi-level pore structure also reduce the aggregation of the fixed enzyme, prevent subunit dissociation and enzyme autolysis or hydrolysis, and also improve the enzyme activity stability of the fixed enzyme; finally, the present invention adopts a support structure with micron-sized pores combined with a load of nano-sized pores, and simultaneously realizes the effective fixation of the composite enzyme and increases the effective contact area between the composite enzyme and the continuously flowing reaction solution, thereby, the porous composite enzyme fixed electrode provided by the present invention can have an efficient enzyme reaction efficiency under continuous flow conditions. At the same time, the present invention only needs to realize the clean regeneration of coenzymes through electric energy and light energy, greatly reducing the use of expensive coenzymes, thereby reducing the synthesis cost.

[0024] The present invention provides a continuous flow enzyme electrocatalytic reaction system, comprising a cathode fixed shell 2, a first waterproof membrane 10, a cathode electrode sheet 3, a second waterproof membrane 12, a cathode reaction chamber 13, a third waterproof membrane 14, an ion exchange membrane 5, a fourth waterproof membrane 15, an anode reaction chamber 16, a fifth waterproof membrane 6, an anode electrode sheet 17, a sixth waterproof membrane 18 and an anode fixed shell 7, which are fixedly connected in sequence; the cathode reaction chamber 13 or the anode reaction chamber 16 is provided with a porous composite enzyme fixed electrode described in the above technical solution or a porous composite enzyme fixed electrode prepared by the preparation method described in the above technical solution. The continuous flow enzyme electrocatalytic reaction system provided by the present invention adopts the porous composite enzyme fixed electrode described in the above technical solution to not only realize the efficient and stable fixation of multiple enzymes; at the same time, the continuously flowing reaction system can destroy the mass transfer boundary layer on the surface of the enzyme fixed electrode, promote the diffusion of reactants to the solid-liquid reaction interface, and at the same time, take away the generated reactants under the flow condition, avoid occupying the active sites on the electrode surface, and promote the system reduction reaction to proceed in the forward direction. Therefore, the continuous flow enzyme electrocatalytic reaction system provided by the present invention has a higher reaction flux, can realize continuous production, and is more suitable for large-scale industrial applications.

[0025] At the same time, the continuous flow enzyme electrocatalytic reaction system provided by the present invention adopts a modular design in structure, so that a single module can be replaced quickly and easily; at the same time, the entire continuous flow enzyme electrocatalytic reaction system has the characteristics of good air tightness, small reaction system impedance, and small energy loss in the reactor. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A schematic diagram of the structure of the continuous flow enzyme electrocatalytic reaction system provided by the present invention;

[0027] Figure 1 In the figure: 1 is a reinforced screw valve, 2 is a cathode fixed shell, 3 is a cathode electrode sheet, 4 is a reference electrode insertion port, 5 is an ion exchange membrane, 6 is a fifth waterproof membrane, 7 is an anode fixed shell, 8 is a fixing screw, 9 is an anti-short circuit silicone sleeve, 10 is a first waterproof membrane, 11 is a reference electrode insertion port fixing screw, 12 is a second waterproof membrane, 13 is a cathode reaction chamber, 14 is a third waterproof membrane, 15 is a fourth waterproof membrane, 16 is an anode reaction chamber, 17 is an anode electrode sheet, 18 is a sixth waterproof membrane, and 19 is a reinforced screw valve gasket;

[0028] Figure 2 This is an electron microscope photo of a porous composite enzyme-immobilized electrode prepared in an embodiment of the present invention;

[0029] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of α-ketoglutaric acid (reaction substrate) used in the examples of the present invention;

[0030] Figure 4The hydrogen nuclear magnetic resonance spectrum of sodium L-glutamate (reaction product) prepared in the embodiment of the present invention;

[0031] Figure 5 This is a current-reaction time variation curve when preparing sodium L-glutamate in Example 1 of the present invention, wherein the ordinate is the current and the abscissa is the reaction time. DETAILED DESCRIPTION

[0032] The present invention provides a porous composite enzyme fixed electrode, comprising a foamed metal, a porous conductive coating arranged on the surface of the foamed metal; and a composite enzyme loaded in the pore structure of the porous conductive coating; the pore structure of the foamed metal is a micrometer-level pore structure, the pore structure of the porous conductive coating is a nanometer-level pore structure, and the porous conductive coating is formed by nano-conductive fillers; the composite enzyme comprises coenzyme reductase, coenzyme and oxidoreductase.

[0033] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0034] The porous composite enzyme immobilization electrode provided by the present invention comprises a foamed metal, wherein the pore structure of the foamed metal is a micron-scale pore structure.

[0035] In the present invention, the foamed metal is preferably foamed nickel.

[0036] In the present invention, the foamed metal is preferably in the form of blocks or particles.

[0037] The present invention has no special requirements on the source of the foamed metal, and commercially available products can be purchased.

[0038] In the present invention, the average pore size of the foamed metal is preferably 20 to 50 μm, more preferably 25 to 45 μm; the porosity is preferably 40 to 90%, more preferably 45 to 85%, and further preferably 50 to 80%.

[0039] In the present invention, the foamed metal serves as a support for the porous composite enzyme fixed electrode, and its porous structure is not only conducive to the loading of the composite enzyme during the preparation process, but also facilitates the contact and reaction between the reaction liquid and the fixed enzyme under continuous flow conditions, thereby improving the reaction efficiency.

[0040] The porous composite enzyme immobilization electrode provided by the present invention comprises a porous conductive coating arranged on the surface of the foamed metal. The pore structure of the porous conductive coating is a nanoscale pore structure, and the porous conductive coating is formed by nano conductive fillers.

[0041] In the present invention, when the foamed metal is in a fast state, the size of the foamed metal is preferably such that it can be inserted into the reaction cavity provided in the cathode reaction chamber 13 or the anode reaction chamber 16 , and matches the reaction cavity provided in the cathode reaction chamber 13 or the anode reaction chamber 16 .

[0042] In the present invention, the thickness of the porous conductive coating is preferably 1 to 3 μm, more preferably 1.5 to 2.5 μm.

[0043] In the present invention, the average pore size of the porous conductive coating is preferably 20 to 80 nm, more preferably 30 to 50 nm; the porosity is preferably 20 to 50%.

[0044] In the present invention, the nano conductive filler preferably includes one or more of nano silver, nano gold, nano indium tin oxide, graphene and carbon nanotube.

[0045] In the present invention, the nano conductive filler also includes other nano-sized materials with conductor or semiconductor properties.

[0046] In the present invention, the porous conductive coating serves as a carrier of the complex enzyme, and the complex enzyme is loaded in the nanoscale pore structure of the porous conductive coating. Since the size of the nanoscale structure is small, the complex enzyme can be effectively fixed.

[0047] The porous composite enzyme fixed electrode provided by the present invention comprises a composite enzyme loaded in the pore structure of the porous conductive coating, wherein the composite enzyme comprises coenzyme reductase, coenzyme and oxidoreductase.

[0048] In the present invention, the coenzyme reductase is preferably ferredoxin-NADP+ reductase (FNR).

[0049] In the present invention, the coenzyme includes one or more of adenosine triphosphate (ATP), reduced coenzyme II (NAD) and reduced coenzyme I (NADP).

[0050] In the present invention, the oxidoreductase is preferably selected according to the requirements of the reaction. In a specific embodiment of the present invention, the oxidoreductase is specifically glutamate dehydrogenase.

[0051] In the present invention, the molar ratio of the coenzyme reductase, the coenzyme and the oxidoreductase is preferably 1:0.0001:10.

[0052] In the present invention, the number of the complex enzyme is preferably 1 to 10.

[0053] The present invention does not need to add expensive coenzyme factors during continuous flow reaction.

[0054] The present invention provides a method for preparing the porous composite enzyme-immobilized electrode described in the above technical solution, comprising the following steps:

[0055] (1) impregnating the foamed metal into a conductive filler slurry, pre-calcining the obtained wet blank in a protective atmosphere and then standing to solidify to obtain an initial solidified embryo; the conductive filler slurry comprises a nano conductive filler, an organic solvent, a binder and a dispersant;

[0056] (2) Repeating the steps of impregnation, pre-calcination and standing curing in step (1) on the initially cured embryo until a conductive cured coating is formed on the surface of the foamed metal;

[0057] (3) calcining the foamed metal having a conductive cured coating on the surface in a protective atmosphere to form a porous conductive coating on the surface of the foamed metal;

[0058] (4) Immersing a foamed metal having a porous conductive coating on its surface into a complex enzyme solution to obtain the porous complex enzyme fixed electrode; the complex enzyme solution comprises coenzyme reductase, coenzyme, oxidoreductase and buffer solution.

[0059] The present invention immerses the foamed metal in a conductive filler slurry, pre-calcines the obtained wet blank in a protective atmosphere and then allows it to stand and solidify to obtain an initial solidified embryo body; the conductive filler slurry includes nano conductive fillers, an organic solvent, a binder and a dispersant.

[0060] In the present invention, the organic solvent preferably includes one or more of acetone, terpinene and N,N-dimethylformamide.

[0061] In the present invention, the binder preferably includes one or more of epoxy resin, methyl hexahydrophthalic anhydride and polyetherimide.

[0062] In the present invention, the dispersant preferably includes polyvinyl pyrrolidone and / or polyvinyl alcohol.

[0063] In the present invention, the mass ratio of the organic solvent, the binder and the dispersant is preferably 1:5:0.1.

[0064] In the present invention, in the conductive filler slurry, the mass percentage of the conductive filler is preferably 1-10%, more preferably 2-8, and most preferably 6%.

[0065] In the present invention, the preparation method of the conductive filler slurry comprises the following steps:

[0066] The organic solvent, the binder and the dispersant are stirred and mixed to obtain an organic slurry;

[0067] The conductive filler and the organic slurry are ultrasonically mixed to obtain the conductive filler slurry.

[0068] In the present invention, the organic solvent, binder and dispersant are stirred and mixed to obtain an organic slurry. The stirring and mixing time in the present invention is preferably 30 minutes.

[0069] After obtaining the organic slurry, the present invention ultrasonically mixes the conductive filler and the organic slurry to obtain the conductive filler slurry. In the present invention, the ultrasonic mixing time is preferably 1 hour; the ultrasonic power of the ultrasonic mixing is preferably 450W. The ultrasonic mixing is performed in an ultrasonic device, and the volume of the ultrasonic device is preferably 15L.

[0070] In the present invention, the immersion temperature is preferably room temperature, and the immersion time is preferably 30 minutes.

[0071] In the present invention, the protective gas is preferably nitrogen.

[0072] In the present invention, the pre-calcination temperature is preferably 350-400°C, more preferably 400°C.

[0073] In the present invention, the holding time of the pre-calcination is preferably 25 minutes.

[0074] In the present invention, the static curing time is preferably 45 minutes; the static curing temperature is preferably room temperature.

[0075] After obtaining the initial solidified embryo body, the present invention repeats the above steps of impregnation-precalcination-stationary solidification on the initial solidified embryo body until a conductive solidified coating is formed on the surface of the foamed metal.

[0076] In the present invention, the thickness of the conductive cured coating is preferably 1 to 3 μm.

[0077] After obtaining the foamed metal with the conductive solidified coating on the surface, the present invention calcines the foamed metal with the conductive solidified coating on the surface in a protective atmosphere to form a porous conductive coating on the surface of the foamed metal.

[0078] In the present invention, the protective gas is preferably nitrogen. The calcination temperature is preferably 600-800° C., more preferably 650-750° C. In the present invention, the calcination holding time is preferably 30 min.

[0079] In the present invention, during the calcination process, the organic solvent is volatilized, and the binder and the dispersant are thermally decomposed, thereby forming a nanoscale pore structure to obtain a porous conductive coating.

[0080] After obtaining the foamed metal with a porous conductive coating on the surface, the present invention immerses the foamed metal with a porous conductive coating on the surface (hereinafter referred to as the first immersion) in a complex enzyme solution to obtain the porous complex enzyme fixed electrode; the complex enzyme solution includes coenzyme reductase, coenzyme, oxidoreductase and buffer solution.

[0081] In the present invention, the first immersion is preferably performed at 4° C. The first immersion is performed under stirring, and the time of the first immersion is preferably 25 minutes.

[0082] In the present invention, the complex enzyme solution comprises coenzyme reductase, coenzyme, oxidoreductase and buffer solution; the buffer solution is preferably a TAPS buffer solution.

[0083] In the present invention, in the complex enzyme solution, the enzyme activity of the coenzyme reductase is preferably ≥20 U / mg; the enzyme activity of the coenzyme is preferably ≥20 U / mg; and the enzyme activity of the oxidoreductase is preferably ≥10 U / mg.

[0084] The present invention provides a continuous flow enzyme electrocatalytic reaction system, comprising a cathode fixed shell 2, a first waterproof membrane 10, a cathode electrode sheet 3, a second waterproof membrane 12, a cathode reaction chamber 13, a third waterproof membrane 14, an ion exchange membrane 5, a fourth waterproof membrane 15, an anode reaction chamber 16, a fifth waterproof membrane 6, an anode electrode sheet 17, a sixth waterproof membrane 18 and an anode fixed shell 7, which are fixedly connected in sequence; the cathode reaction chamber 13 or the anode reaction chamber 16 is provided with the porous composite enzyme fixed electrode described in the above technical solution or the porous composite enzyme fixed electrode prepared by the preparation method described in the above technical solution.

[0085] like Figure 1 As shown:

[0086] The continuous flow enzyme electrocatalytic reaction system provided by the present invention comprises a cathode fixing shell 2. In the present invention, the cathode fixing shell 2 is used to protect the cathode electrode sheet 3.

[0087] The continuous flow enzyme electrocatalytic reaction system provided by the present invention comprises a first waterproof membrane 10 fixedly connected to the cathode fixed housing 2. In the present invention, the material of the first waterproof membrane 10 is preferably silica gel. The function of the first waterproof membrane 10 is to prevent liquid leakage between the cathode fixed housing 2 and the cathode electrode sheet 3.

[0088] The continuous flow enzyme electrocatalytic reaction system provided by the present invention comprises a cathode electrode sheet 3 fixedly connected to the first waterproof membrane 10. In the present invention, the cathode electrode sheet is used for connecting to an external power source to perform an electrocatalytic reaction.

[0089] The continuous flow enzyme electrocatalytic reaction system provided by the present invention comprises a second waterproof membrane 12 fixedly connected to the cathode electrode sheet 3. In the present invention, the material of the second waterproof membrane 12 is preferably silica gel. The function of the second waterproof membrane 12 is to prevent liquid leakage between the cathode electrode sheet 3 and the cathode reaction chamber 13.

[0090] The continuous flow enzyme electrocatalytic reaction system provided by the present invention comprises a cathode reaction chamber 13 fixedly connected to the second waterproof membrane 12 .

[0091] As one or more embodiments of the present invention, in the present invention, a reaction chamber is provided in the cathode reaction chamber 13, and the reaction chamber is used for performing enzyme electrocatalytic reaction. In the present invention, the cathode reaction chamber 13 is provided with a reaction chamber having the porous enzyme immobilization electrode described in the above technical solution built therein.

[0092] As one or more embodiments of the present invention, the cathode reaction chamber 13 is provided with a reference electrode interface 4. In the present invention, the reference electrode interface 4 is used to connect an external reference electrode. In the present invention, the reference electrode is inserted into the reaction cavity of the cathode reaction chamber 13 through the reference electrode interface 4, providing data support for real-time monitoring and intervention of the reaction in the cathode reaction chamber 13.

[0093] As one or more embodiments of the present invention, the cathode reaction chamber 13 is provided with a reference electrode insertion port fixing screw 11 matched with the reference electrode interface 4. In the present invention, when the reference electrode interface 4 is not in use, the reference electrode insertion port fixing screw 11 is inserted into the reference electrode interface 4 to seal the reference electrode interface 4.

[0094] As one or more embodiments of the present invention, the cathode reaction chamber 13 is provided with one reaction liquid inlet and one reaction liquid outlet.

[0095] As one or more embodiments of the present invention, the cathode reaction chamber 13 is further provided with a spare reaction liquid inlet and a spare reaction liquid outlet.

[0096] As one or more embodiments of the present invention, the reaction cavity of the cathode reaction chamber 13 is provided with the porous composite enzyme fixed electrode described in the above technical solution or the porous composite enzyme fixed electrode prepared by the preparation method described in the above technical solution. In the present invention, when the reaction raw material liquid enters from the inlet of the cathode reaction chamber 13, it contacts the porous composite enzyme fixed electrode in the reaction cavity of the cathode reaction chamber 13, and an enzymatic electrocatalytic reaction occurs under the condition of an external power supply to obtain a reaction product.

[0097] The continuous flow enzyme electrocatalytic reaction system provided by the present invention comprises a third waterproof membrane 14 fixedly connected to the cathode reaction chamber 13. In the present invention, the material of the third waterproof membrane 14 is preferably silica gel. The function of the third waterproof membrane 14 is to prevent liquid leakage between the cathode reaction chamber 13 and the ion exchange membrane 5.

[0098] The continuous flow enzyme electrocatalytic reaction system provided by the present invention comprises an ion exchange membrane 5 fixedly connected to the third waterproof membrane 14. In the present invention, the cathode reaction chamber 13 and the anode reaction chamber 16 are separated by the ion exchange membrane 5, and different electrolytes can be added to the cathode reaction chamber 13 and the anode reaction chamber 16 according to different types of reactions without interfering with each other.

[0099] The continuous flow enzyme electrocatalytic reaction system provided by the present invention comprises a fourth waterproof membrane 15 fixedly connected to the ion exchange membrane 5. In the present invention, the material of the fourth waterproof membrane 15 is preferably silica gel. The function of the fourth waterproof membrane 15 is to prevent liquid leakage between the ion exchange membrane 5 and the anode reaction chamber 16.

[0100] The continuous flow enzyme electrocatalytic reaction system provided by the present invention comprises an anode reaction chamber 16 fixedly connected to the fourth waterproof membrane 15 .

[0101] As one or more embodiments of the present invention, in the present invention, a reaction chamber is provided in the anode reaction chamber 16, and the reaction chamber is used for performing enzyme electrocatalytic reaction.

[0102] As one or more embodiments of the present invention, the anode reaction chamber 16 is provided with one reaction liquid inlet and one reaction liquid outlet.

[0103] As one or more embodiments of the present invention, the reaction cavity of the anode reaction chamber 16 is provided with the porous composite enzyme fixed electrode described in the above technical solution or the porous composite enzyme fixed electrode prepared by the preparation method described in the above technical solution. In the present invention, when the reaction raw material liquid enters from the inlet of the anode reaction chamber 16, it contacts the porous composite enzyme fixed electrode in the reaction cavity of the anode reaction chamber 16, and an enzyme-catalyzed electrocatalytic reaction occurs under the condition of an external power supply to obtain a reaction product.

[0104] In the present invention, when the reaction chamber of the cathode reaction chamber 13 is provided with the porous complex enzyme fixed electrode described in the above technical scheme or the porous complex enzyme fixed electrode prepared by the preparation method described in the above technical scheme, the reaction chamber of the anode reaction chamber 16 is passed through a buffer solution that can provide electrons to the cathode reaction chamber 13.

[0105] In the present invention, the ratio of the flow rate of the reaction solution in the cathode reaction chamber 13 to the flow rate of the buffer solution in the anode reaction chamber 16 is preferably 1 to 200:1.

[0106] The continuous flow enzyme electrocatalytic reaction system provided by the present invention comprises a fifth waterproof membrane 6 fixedly connected to the anode reaction chamber 16. In the present invention, the material of the fifth waterproof membrane 6 is preferably silica gel. The function of the fifth waterproof membrane 6 is to prevent liquid leakage between the anode reaction chamber 16 and the anode electrode sheet 17.

[0107] The continuous flow enzyme electrocatalytic reaction system provided by the present invention comprises an anode electrode sheet 17 fixedly connected to the fifth waterproof membrane 6. In the present invention, the anode electrode sheet 17 is used for an external power supply, and forms a closed-circuit current with the cathode electrode sheet 3 to provide electric energy for the continuous flow enzyme electrocatalytic reaction system.

[0108] The continuous flow enzyme electrocatalytic reaction system provided by the present invention comprises a sixth waterproof membrane 18 fixedly connected to the anode electrode sheet 17. In the present invention, the material of the sixth waterproof membrane 18 is preferably silica gel. The function of the sixth waterproof membrane 18 is to prevent liquid leakage between the anode electrode sheet 17 and the anode fixed housing 7.

[0109] The continuous flow enzyme electrocatalytic reaction system provided by the present invention comprises an anode fixed housing 7 fixedly connected to the sixth waterproof membrane 18. In the present invention, the anode fixed housing 7 is used to protect the anode electrode sheet 7.

[0110] As one or more specific embodiments of the present invention, the cathode fixed housing 2, the cathode reaction chamber 13, the anode reaction chamber 16, and the anode fixed housing 7 are respectively made of transparent materials.

[0111] As one or more specific embodiments of the present invention, the cathode fixed housing 2, the cathode reaction chamber 13, the anode reaction chamber 16, and the anode fixed housing 7 are made of transparent quartz respectively.

[0112] As one or more specific embodiments of the present invention, the cathode fixed housing 2, cathode reaction chamber 13, anode reaction chamber 16, and anode fixed housing 7 are respectively made of transparent quartz, which can not only provide the light transmission requirements for photocatalysis, but also make the reaction process clearly visible.

[0113] In the present invention, the energy provided by photocatalysis can be used for the regeneration of ATP.

[0114] When the cathode reaction chamber 13 and the anode reaction chamber 16 are preferably made of transparent materials, the continuous flow enzyme electrocatalytic reaction system can also perform enzymatic photocatalytic reactions.

[0115] As one or more specific embodiments of the present invention, the cathode fixed shell 2, the first waterproof membrane 10, the cathode electrode sheet 3, the second waterproof membrane 12, the cathode reaction chamber 13, the third waterproof membrane 14, the ion exchange membrane 5, the fourth waterproof membrane 15, the anode reaction chamber 16, the fifth waterproof membrane 6, the anode electrode sheet 17, the sixth waterproof membrane 18 and the anode fixed shell 7 are respectively provided with screw openings; the number of the screw openings is 4 respectively.

[0116] The continuous flow enzyme electrocatalytic reaction system provided by the present invention preferably also includes a fixing screw 8. In the present invention, the fixing screw is used to simultaneously penetrate the cathode fixed housing 2, the first waterproof membrane 10, the cathode electrode sheet 3, the second waterproof membrane 12, the cathode reaction chamber 13, the third waterproof membrane 14, the ion exchange membrane 5, the fourth waterproof membrane 15, the anode reaction chamber 16, the fifth waterproof membrane 6, the anode electrode sheet 17, the sixth waterproof membrane 18 and the screw opening on the anode fixed housing 7, and is fixed by the reinforcement screw valve 1.

[0117] As one or more embodiments of the present invention, the number of the fixing screws 8 is four.

[0118] As one or more embodiments of the present invention, the fixing screw 8 that passes through the cathode fixed shell 2, the first waterproof membrane 10, the cathode electrode sheet 3, the second waterproof membrane 12, the cathode reaction chamber 13, the third waterproof membrane 14, the ion exchange membrane 5, the fourth waterproof membrane 15, the anode reaction chamber 16, the fifth waterproof membrane 6, the anode electrode sheet 17, the sixth waterproof membrane 18 and the anode fixed shell 7 is provided with an anti-short circuit silicone layer.

[0119] As one or more embodiments of the present invention, a reinforcing screw valve gasket 19 is provided at the fixed connection between the fixing screw 8 and the reinforcing screw valve 1 .

[0120] The continuous flow enzyme electrocatalytic reaction system provided by the present invention preferably further comprises a raw material liquid storage tank. The raw material liquid storage tank is used to store the reaction liquid.

[0121] As one or more embodiments of the present invention, a delivery pump is provided on the pipeline connecting the raw liquid storage tank and the liquid inlet of the cathode reaction chamber 13 or the anode reaction chamber 16 .

[0122] As one or more embodiments of the present invention, the delivery pump is a peristaltic pump.

[0123] The continuous flow enzyme electrocatalytic reaction system provided by the present invention preferably further comprises a product collection tank, which is used to collect the product solution obtained after the reaction.

[0124] In the present invention, when the continuous flow enzyme electrocatalytic reaction system is in operation, the present invention preferably uses the Ivium potentiometry and the chronoamperometry to start and control the system.

[0125] In the present invention, the liquid outlet of the continuous flow enzyme electrocatalytic reaction system can be connected back to the raw liquid storage tank to form a closed loop system, or can be connected to another continuous flow enzyme electrocatalytic reaction system for further reaction. In the present invention, if the conversion rate of the reaction is very high, the liquid outlet of the continuous flow enzyme electrocatalytic reaction system is directly connected to the product collection tank.

[0126] In the present invention, the continuous flow enzyme electrocatalytic reaction system is preferably designed as a flow-through reactor, a ring reactor or a continuous stirred tank reactor (CSTR), and the specific design depends on the requirements.

[0127] In a specific embodiment of the present invention, the continuous flow enzyme electrocatalytic reaction system is a ring reactor.

[0128] The continuous flow enzyme electrocatalytic reaction system provided by the present invention has the following characteristics:

[0129] (1) The present invention can achieve clean regeneration of ATP / NADPH / NADH through electrical energy and / or light energy, greatly reducing the use of expensive coenzymes, thereby reducing synthesis costs.

[0130] (2) The continuous flow enzyme electrocatalytic reaction system provided by the present invention has good air tightness, low impedance of the reaction system, small reactor and low energy loss; and the reaction system adopts a functional modular design, so that a single functional module can be replaced quickly and easily.

[0131] (3) The continuous flow enzyme electrocatalytic reaction system provided by the present invention can achieve one temperature for the reaction chamber and one temperature for the reaction liquid collector. For example, when the continuous flow enzyme electrocatalytic reaction system provided by the present invention is used, the reaction temperature in the reaction chamber of the cathode reaction chamber 13 can rise to 60°C. At this temperature, not only the enzyme reaction rate is accelerated, but also the solubility of the reactants is often greatly improved. At the same time, in order to avoid the decomposition of the product at high temperature, the outflowing product can be quickly cooled and recovered to the reaction liquid collector.

[0132] The present invention provides the use of the porous composite enzyme fixed electrode described in the above technical scheme or the porous composite enzyme fixed electrode prepared by the preparation method described in the above technical scheme or the continuous flow enzyme electrocatalytic reaction system described in the above technical scheme in enzyme electrocatalytic synthesis of chemical substances.

[0133] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0134] Example 1

[0135] (1) A solvent (acetone), a binder (epoxy resin) and a dispersant (polyvinyl pyrrolidone) are mixed and stirred at a corresponding mass ratio of 1:5:0.1 for 30 minutes to form a stable organic slurry; a conductive filler (nanosilver) and the organic slurry are mixed and ultrasonicated for 1 hour at an ultrasonic power of 450 W to form a stable and uniform conductive electrode slurry, wherein the mass percentage of nanosilver in the conductive electrode slurry is 6%.

[0136] (2) The nickel foam (average pore size of 50 μm, porosity of 20%) is infiltrated into the conductive electrode slurry prepared in step (1), and the wet blank is taken out after soaking for 30 minutes, pre-fired at 400°C for 25 minutes, and statically cured for 45 minutes, and then put into the conductive electrode slurry again to repeat the soaking-pre-fire-static curing process until the slurry thickness reaches 3 μm, and then fired at 600°C to volatilize the acetone, decompose the epoxy resin and polyvinyl pyrrolidone by heat, and form a porous conductive coating on the surface of the nickel foam. The average pore size of the porous conductive coating is 20 nm, and the porosity is 50%. The electron microscope photo is shown in FIG. Figure 2 shown.

[0137] (3) NADP reductase (ferredoxin-NADP+ reductase), oxidized coenzyme II (NADPH, purchased from Sigma-Aldrich) and glutamate dehydrogenase (GLDH) were added to a TAPS buffer solution at a molar ratio of 1:0.0001:10 and mixed evenly to obtain a composite enzyme solution; then the nickel foam with a porous conductive coating obtained in step (2) was immersed in the composite enzyme solution, stirred at an ambient temperature of 4°C for 25 minutes, and a porous composite enzyme fixed electrode loaded with enzyme was obtained.

[0138] Example 2

[0139] according to Figure 1 The schematic diagram of the continuous flow enzyme electrocatalytic reaction system shown in the figure is as follows: the porous composite enzyme fixed electrode prepared in Example 1 is placed in the reaction cavity of the cathode reaction chamber 13 and locked; Figure 1 The structural schematic diagram of the continuous flow enzyme electrocatalytic reaction system was assembled.

[0140] α-ketoglutaric acid and ammonium chloride (providing ammonium ions) are dissolved in a TAPS buffer solution to obtain a reaction raw material solution; wherein the mass concentration of α-ketoglutaric acid is 40 mmol / L; the mass concentration of ammonium chloride is 10 g / mL; 50 mL of the reaction raw material solution is stored in a reaction raw material solution storage tank, and the liquid outlet of the reaction raw material solution storage tank is connected to a plastic pipe (PVC pipe) through a peristaltic pump Figure 1The liquid inlet of the cathode reaction chamber 13 of the continuous flow enzyme electrocatalytic reaction system is assembled, and after the liquid flows out from the liquid outlet of the cathode reaction chamber 13, it flows back to the reaction raw material liquid storage tank to form a closed-loop system.

[0141] The continuous flow enzyme electrocatalytic reaction system is connected to the Ivium electrochemical workstation, and the reaction is started and controlled using the chronoamperometry. Glutamate dehydrogenase (GLDH), NADP reductase and oxidized coenzyme II are loaded on the porous fixed enzyme electrode prepared in Example 1 of the potentiometer, and a voltage (-0.75V, a reference electrode is silver / silver chloride, in 3.5mol / L potassium chloride) is applied to the working electrode using a computer-controlled potentiometer, and a peristaltic pump is turned on to inject the reaction raw material liquid containing α-ketoglutaric acid and ammonium chloride in the storage tank into the continuous flow enzyme electrocatalytic reaction system. With the flow of the solution (flow rate is 2mL / s), and under the applied voltage, the coenzyme can be repeatedly regenerated on the porous composite enzyme fixed electrode, and the reaction is started thereupon. The chronoamperometry monitors the reaction rate in real time, and displays the reaction progress on the computer screen. After 14 hours, the reactant (α-ketoglutaric acid) is completely converted into the product sodium L-glutamate with the help of GLDH and coenzyme regeneration enzyme, with a yield of 99.8%.

[0142] Depend on Figure 5 It can be seen that the reaction current reaches 15mA at the beginning of the reaction, and the reaction reaches equilibrium (the current is close to 0) in about 14 hours. Figure 5 This is a schematic diagram of electrochemical workstation detection, where the horizontal and vertical axes correspond to time and current intensity respectively.

[0143] Repeated experiments have verified that the porous composite enzyme electrode in the continuous flow enzyme electrocatalytic reaction system provided by the present invention can be reused, and the reaction efficiency remains basically unchanged after 5 repetitions.

[0144] The continuous flow enzyme electrocatalytic reaction system provided by the invention is simple, efficient and easy to use in synthesizing compounds.

[0145] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. A method for preparing a porous composite enzyme-immobilized electrode, characterized in that: The porous complex enzyme fixed electrode comprises a foamed metal, a porous conductive coating arranged on the surface of the foamed metal; and a complex enzyme loaded in the pore structure of the porous conductive coating; the pore structure of the foamed metal is a micrometer-level pore structure, the pore structure of the porous conductive coating is a nanometer-level pore structure, and the porous conductive coating is formed by nano-conductive fillers; the complex enzyme comprises coenzyme reductase, coenzyme and oxidoreductase; the nano-conductive fillers comprise one or more of nano-silver, nano-gold, nano-indium tin oxide, graphene and carbon nanotubes; the foamed metal is foamed nickel; The preparation method of the porous composite enzyme-immobilized electrode comprises the following steps: (1) impregnating the foamed metal into a conductive filler slurry, pre-calcining the obtained wet blank in a protective atmosphere and then standing to solidify to obtain an initial solidified embryo; the conductive filler slurry comprises a nano conductive filler, an organic solvent, a binder and a dispersant; The binder includes one or more of epoxy resin, methyl hexahydrophthalic anhydride and polyetherimide; The dispersant includes polyvinyl pyrrolidone and / or polyvinyl alcohol; The organic solvent includes one or more of acetone, terpinene and N,N-dimethylformamide; (2) Repeating the steps of impregnation, pre-calcination and standing curing in step (1) on the initial cured embryo until a conductive cured coating is formed on the surface of the foamed metal; (3) calcining the foamed metal having a conductive cured coating on the surface in a protective atmosphere to form a porous conductive coating on the surface of the foamed metal; (4) Immersing a foamed metal having a porous conductive coating on its surface into a complex enzyme solution to obtain the porous complex enzyme fixed electrode; the complex enzyme solution comprises coenzyme reductase, coenzyme, oxidoreductase and buffer solution.

2. The method for preparing the porous composite enzyme-immobilized electrode according to claim 1, characterized in that: The average pore size of the pore structure of the foamed metal is 20-50 μm, and the porosity is 40-90%.

3. The method for preparing the porous composite enzyme-immobilized electrode according to claim 1, characterized in that: The thickness of the porous conductive coating is 1-3 μm; the average pore size of the pore structure of the porous conductive coating is 20-80 nm, and the porosity is 20-50%.

4. The method for preparing the porous composite enzyme-immobilized electrode according to claim 1, characterized in that: The coenzyme includes one or more of adenosine triphosphate, reduced coenzyme II and reduced coenzyme I.

5. The preparation method according to claim 1, characterized in that: The temperature of the pre-calcination is 250-400°C; the temperature of the calcination is 600-800°C.

6. A continuous flow enzyme electrocatalytic reaction system, characterized in that: The invention comprises a cathode fixed shell (2), a first waterproof membrane (10), a cathode electrode sheet (3), a second waterproof membrane (12), a cathode reaction chamber (13), a third waterproof membrane (14), an ion exchange membrane (5), a fourth waterproof membrane (15), an anode reaction chamber (16), a fifth waterproof membrane (6), an anode electrode sheet (17), a sixth waterproof membrane (18) and an anode fixed shell (7) which are fixedly connected in sequence; the cathode reaction chamber (13) or the anode reaction chamber (16) is provided with a porous composite enzyme fixed electrode prepared by the method for preparing a porous composite enzyme fixed electrode according to any one of claims 1 to 5.

7. Use of the porous composite enzyme immobilized electrode prepared by the preparation method of the porous composite enzyme immobilized electrode according to any one of claims 1 to 5 or the continuous flow enzyme electrocatalytic reaction system according to claim 6 in enzyme electrocatalytic synthesis of chemical substances.

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