A method for synthesizing NADPH by electrocatalysis of immobilized enzyme

Abstract

The present invention belongs to the field of biopharmaceutical technology, and specifically relates to a method for electrically promoting the synthesis of NADPH by immobilized enzyme catalysis. The present invention adopts a three-electrode system to + The buffer solution is used as the reaction solution, and the immobilized enzyme electrode is used as the working electrode to perform an electrocatalytic enzyme reduction reaction to obtain the NADPH; the immobilized enzyme electrode includes a semiconductor substrate, a porous conductive filler coating disposed on the surface of the semiconductor substrate, and a ferredoxin-NADP+ reductase loaded in the pore structure of the porous conductive filler coating. The present invention adopts a three-electrode system. Under an electric environment, NADP + As a substrate, NADPH is synthesized by catalysis of an immobilized enzyme electrode loaded with ferredoxin-NADP+ reductase (FNR). The method provided by the invention has the characteristics of high yield, high purity, simple synthesis route, low cost, and environmental friendliness.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a method for synthesizing NADPH by electrocatalytic immobilized enzyme. Background Art

[0002] NADPH (reduced nicotinamide adenine dinucleotide phosphate) is a coenzyme found in living cells. NADPH is synthesized via the pentose phosphate pathway, a metabolic pathway that converts glucose into NADPH and ribose-5-phosphate.

[0003] Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for many reductive biosynthetic processes, such as fatty acid synthesis, cholesterol synthesis, bile acid synthesis, steroid hormone synthesis, and deoxynucleotide synthesis. NADPH is also used in the process of cell protection against redox stress by providing reducing equivalents for antioxidants such as glutathione and thioredoxin. NADPH can participate in the regeneration of glutathione, which is a key antioxidant in organisms.

[0004] Currently, NADPH is used in the biotechnology industry and can be used to produce chemicals. In chemical production, NADPH is often used to produce chiral intermediates for drugs. These chiral intermediates are then further processed to produce chiral drugs with high purity and efficacy. For example, NADPH is used to synthesize (S)-naproxen, a nonsteroidal anti-inflammatory drug (NSAID) used to treat pain and inflammation. The (S)-enantiomer of naproxen is more active than the (R)-enantiomer, making it the preferred form for therapeutic use. The synthesis of (S)-naproxen involves the use of NADPH-dependent ketoreductase to reduce the keto group in the precursor compound (S)-2'-methoxy-2-methylpropiophenone. This ketoreductase catalyzes the stereospecific reduction of the keto group to produce (S)-2'-methoxy-2-methylpropionic acid methyl ester with high enantioselectivity. The intermediate is then converted to (S)-naproxen through a series of chemical reactions.

[0005] The use of NADPH-dependent enzymes in the synthesis of chiral drugs can produce high-purity enantiomers with minimal side effects. This approach is becoming increasingly popular in the pharmaceutical industry because it offers a more sustainable and efficient alternative to traditional chemical synthesis methods.

[0006] NADPH is NADP +The reduced form of NADPH. Currently, the methods for synthesizing NADPH can be divided into biological fermentation and chemical synthesis. The biological fermentation method is to separate and purify various products of microorganisms by using biological fermentation technology or other microbial culture technology. The biological fermentation method has the disadvantages of huge raw material input, low yield, high energy consumption, and high cost of process separation and purification, which leads to excessively high product prices and limits the widespread application of NADPH. The traditional chemical synthesis method obtains NADPH through multi-step organic synthesis, but this method has many reaction steps, harsh reaction conditions, requires toxic and harmful reagents, and poor product selectivity, resulting in low product purity and yield, which limits the large-scale production and application of NADPH.

[0007] Enzyme-catalyzed synthesis of NADPH is a highly efficient and selective synthesis reaction route. The product has specific selectivity, mild reaction conditions, high yield, and friendly reaction reagent conditions. Currently, the most commonly used enzymatic synthesis of NADPH is GDH and FDH. GDH has high activity, but requires additional glucose as a substrate. The byproduct gluconic acid can easily lead to enzyme inactivation, resulting in a decrease in yield, and the subsequent separation and purification of NADPH also faces huge challenges. On the other hand, FDH is convenient for the subsequent separation and purification of the product NADPH, but carbon dioxide is produced as a byproduct during the production process, which pollutes the environment.

[0008] In summary, the current enzymatic production of NADPH has the problems of low yield and serious pollution. Summary of the invention

[0009] The object of the present invention is to provide a method for synthesizing NADPH by electrocatalytic immobilized enzyme. The method provided by the present invention has the characteristics of high yield, high purity, simple synthesis route, low cost and environmental friendliness.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] The present invention provides a method for synthesizing NADPH by electrocatalytic immobilized enzyme, comprising the following steps:

[0012] A three-electrode system was used to + The buffer solution is used as the reaction solution, and the immobilized enzyme electrode is used as the working electrode to perform an electro-enzyme catalytic reduction reaction to obtain the NADPH;

[0013] The immobilized enzyme electrode comprises a semiconductor substrate, a porous conductive filler coating arranged on the surface of the semiconductor substrate, and a ferredoxin-NADP+ reductase loaded in the pore structure of the porous conductive filler coating.

[0014] Preferably, the mass ratio of the ferredoxin-NADP+ reductase to the porous conductive filler coating is 0.01-0.02:1.

[0015] Preferably, NADP + The molar ratio of the precipitate to the ferredoxin-NADP+ reductase is 90,000 to 100,000:1.

[0016] Preferably, the conductive filler is indium tin oxide or a mixture of indium tin oxide and antimony tin oxide; the pore size of the porous conductive filler coating is 10 to 100 nm.

[0017] Preferably, the semiconductor substrate is made of titanium dioxide.

[0018] Preferably, the reference electrode in the three-electrode system is Ag / AgCl, and the voltage of the working electrode is -0.75 to -0.8V.

[0019] Preferably, the temperature of the electroenzyme-catalyzed reduction reaction is 5 to 45° C., the time is 4 to 24 hours, and the pH value is 6.8 to 8.2.

[0020] Preferably, the preparation method of the immobilized enzyme electrode comprises the following steps:

[0021] A semiconductor substrate is used as a negative electrode, an organic solution containing a conductive filler is used as an electrolyte, and a porous conductive filler coating is formed on the surface of the semiconductor substrate by an electrophoresis treatment method;

[0022] The buffer solution containing ferredoxin-NADP+ reductase is coated on the porous conductive filler coating, and the immobilized enzyme electrode is obtained after standing at a temperature below 25° C.

[0023] Preferably, when the conductive filler is a mixture of indium tin oxide and antimony tin oxide, in the organic solution containing the conductive filler, the mass concentration of indium tin oxide is 0.9-1.5 mg / mL, and the mass concentration of antimony tin oxide is 0.1-0.2 mg / mL.

[0024] Preferably, the voltage of the electrophoresis treatment is 10 to 15 V, and the time is 7 to 15 min.

[0025] The present invention provides a method for synthesizing NADPH by electrocatalysis of immobilized enzyme, comprising the following steps: using a three-electrode system to +The buffer solution is used as the reaction solution, and the immobilized enzyme electrode is used as the working electrode to perform an electrocatalytic enzyme reduction reaction to obtain the NADPH; the immobilized enzyme electrode includes a semiconductor substrate, a porous conductive filler coating disposed on the surface of the semiconductor substrate, and a ferredoxin-NADP+ reductase loaded in the pore structure of the porous conductive filler coating. The present invention adopts a three-electrode system. Under an electric environment, NADP + As a substrate, NADPH is synthesized by catalysis of an immobilized enzyme electrode loaded with ferredoxin-NADP+ reductase (FNR). The method provided by the present invention has the following advantages:

[0026] The method for synthesizing NADPH by electrocatalytic immobilized enzyme provided by the present invention can realize continuous reaction synthesis of NADPH under power-on environment, shorten the production cycle (reaction time is controlled within 24 hours), and improve the purity and yield of NADPH (purity ≥95%, yield ≥85%).

[0027] The present invention uses an immobilized enzyme electrode to synthesize NADPH, and the immobilized enzyme electrode can be used repeatedly, thereby improving the use efficiency of FNR and reducing the production cost. On the other hand, the present invention uses an immobilized enzyme electrode to produce NADPH, so that NADPH is easy to separate, protein residues are avoided, the purification steps are simplified, and the purity of the product is improved.

[0028] The present invention adopts an immobilized enzyme electrode to synthesize NADPH, loads FNR in the pore structure of a porous conductive filler, improves the loading strength of the enzyme, is easy to be applied to a continuous automated reactor, and is conducive to the large-scale production of NADPH.

[0029] The method for synthesizing NADPH by electrocatalytic immobilized enzyme provided by the present invention does not require the addition of other compounds, thus avoiding the introduction of impurities and being beneficial to improving the purity of NADPH. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The NADPH synthesis principle diagram provided by the embodiment of the present invention;

[0031] Figure 2 is a curve diagram showing the change of reaction current over reaction time in Example 1;

[0032] Figure 3 This is an example diagram of the purity of the reaction product prepared in Example 1. DETAILED DESCRIPTION

[0033] The present invention provides a method for synthesizing NADPH by electrocatalytic immobilized enzyme, comprising the following steps:

[0034] A three-electrode system was used to+ The buffer solution is used as the reaction solution, and the immobilized enzyme electrode is used as the working electrode to perform an electro-enzyme catalytic reduction reaction to obtain the NADPH;

[0035] The immobilized enzyme electrode comprises a semiconductor substrate, a porous conductive filler coating arranged on the surface of the semiconductor substrate, and a ferredoxin-NADP+ reductase loaded in the pore structure of the porous conductive filler coating.

[0036] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0037] The method provided by the present invention adopts a three-electrode system, which includes a working electrode, a reference electrode and a counter electrode.

[0038] In the present invention, the working electrode is an immobilized enzyme electrode.

[0039] The immobilized enzyme electrode provided by the present invention comprises a semiconductor substrate. In the present invention, the material of the semiconductor substrate is preferably titanium dioxide. In the present invention, the shape of the semiconductor substrate is sheet-like.

[0040] The immobilized enzyme electrode provided by the present invention comprises a porous conductive filler coating disposed on the surface of the semiconductor substrate. In the present invention, the conductive filler is preferably indium tin oxide or a mixture of indium tin oxide and antimony tin oxide, more preferably a mixture of indium tin oxide (ITO) and antimony tin oxide (ATO), and in the mixture of indium tin oxide and antimony tin oxide, the mass ratio of ITO to ATO is preferably (0.9-1.5):(0.1-0.2), more preferably 9:1.

[0041] In the present invention, the pore size of the porous conductive filler coating is preferably 10 to 100 nm, more preferably 20 to 80 nm.

[0042] In the present invention, the pore size of the porous conductive filler coating is preferably 10 to 100 nm, which can improve the binding strength of the coating with the enzyme when loaded with FNR reductase, enable the obtained immobilized enzyme working electrode to be used repeatedly, and avoid the introduction of new impurities.

[0043] In the present invention, the porous conductive filler coating is preferably disposed on both surfaces of the semiconductor substrate. The thickness of the porous conductive filler coating is preferably 3 to 10 mm.

[0044] The immobilized enzyme electrode provided by the present invention comprises ferredoxin-NADP+ reductase (FNR) loaded in the pore structure of the porous conductive filler coating.

[0045] In the present invention, the mass ratio of the ferredoxin-NADP+ reductase to the porous conductive filler coating is preferably 0.01-0.02:1, more preferably 0.017:1.

[0046] In the present invention, the method for preparing the immobilized enzyme electrode preferably comprises the following steps:

[0047] A semiconductor substrate is used as a negative electrode, an organic solution containing a conductive filler is used as an electrolyte, and a porous conductive filler coating is formed on the surface of the semiconductor substrate by an electrophoresis treatment method;

[0048] A buffer solution containing ferredoxin-NADP+ reductase is coated on the porous conductive filler coating, and the immobilized enzyme electrode is obtained after standing.

[0049] The invention uses a semiconductor substrate as a negative electrode, an organic solution containing a conductive filler as an electrolyte, and adopts an electrophoresis treatment method to form a porous conductive filler coating on the surface of the semiconductor substrate.

[0050] In the present invention, the organic solution containing the conductive filler preferably comprises a conductive filler, an organic solvent and an iodine element. The organic solvent is preferably acetone. The iodine element is used to improve the conductivity of the organic solution containing the conductive filler.

[0051] In the present invention, when the conductive filler is a mixture of indium tin oxide and antimony tin oxide, the mass concentration of indium tin oxide in the organic solution containing the conductive filler is preferably 0.9-1.5 mg / mL, more preferably 0.9 mg / mL; the mass concentration of antimony tin oxide is preferably 0.1-0.2 mg / mL, more preferably 0.1 mg / mL. In the present invention, the mass concentration of iodine in the organic solution containing the conductive filler is preferably 1 mg / mL.

[0052] In the present invention, during the electrophoresis treatment, the present invention preferably uses ITO glass as the positive electrode to form a current closed circuit with the semiconductor substrate, the organic solution containing the conductive filler and the external power supply.

[0053] In the present invention, before the electrophoresis treatment, the organic solution containing the conductive filler is preferably subjected to ultrasonic treatment, the time of the ultrasonic treatment is preferably 30 minutes, and the power of the ultrasonic treatment is preferably 150-500W.

[0054] In the present invention, the voltage of the electrophoresis treatment is preferably 10 to 15 V, more preferably 15 V, and the time is preferably 7 to 15 min.

[0055] In the present invention, the specific implementation method of the electrophoretic treatment is preferably: connect the semiconductor substrate to the negative pole of an external power source and immerse it in an organic solution containing a conductive filler; connect the ITO glass to the positive pole of an external power source and immerse it in an organic solution containing a conductive filler; turn on the external power source to perform the electrophoretic treatment. In the present invention, during the electrophoretic treatment, the present invention preferably performs the electrophoretic treatment on both surfaces of the semiconductor substrate separately, and prepares a porous conductive filler coating on both surfaces of the semiconductor substrate; the time for performing the electrophoretic treatment on each surface of the semiconductor substrate is preferably 7 minutes. In a specific embodiment of the present invention, the present invention preferably turns on the external power source to perform the electrophoretic treatment for 7 minutes, then turns the semiconductor substrate over and connects the external power source, and continues to perform the electrophoretic treatment on the other surface of the semiconductor substrate.

[0056] After forming a porous conductive filler coating on the surface of the semiconductor substrate, the present invention applies a buffer solution containing ferredoxin-NADP+ reductase on the porous conductive filler coating and leaves it to stand at a temperature below 25° C. to obtain the immobilized enzyme electrode.

[0057] In the present invention, the buffer solution containing ferredoxin-NADP+ reductase preferably includes ferredoxin-NADP+ reductase and a buffer solution, and the buffer solution is preferably a buffer solution containing HEPES. The molar concentration of the HEPES buffer solution is preferably 50mmol / L, and the pH value is preferably 7.5. The pH value of the buffer solution containing ferredoxin-NADP+ reductase is preferably 7.5.

[0058] The present invention preferably coats the porous conductive coatings disposed on the two surfaces of the semiconductor substrate with a buffer solution containing ferredoxin-NADP+ reductase. In the present invention, the coating temperature is preferably 4° C. The present invention has no special requirements for the specific implementation process of the coating.

[0059] In the present invention, the standing time is preferably 1 hour.

[0060] In the present invention, the reference electrode is preferably an Ag / AgCl electrode.

[0061] In the present invention, the counter electrode is preferably a platinum sheet.

[0062] In the present invention, the reference electrode is disposed in a NaCl solution, and the molar concentration of the NaCl solution is preferably 0.1 mmol / L.

[0063] In the present invention, the working electrode and the counter electrode are arranged in an electrolyte.

[0064] In the present invention, the reaction solution contains NADP + of buffer solution.

[0065] In the present invention, NADP + The buffer solution preferably includes NADP + and HEPES buffer solution. The molar concentration of the HEPES buffer solution is preferably 50 mmol / L. + The pH value of the buffer solution is preferably 7.5. + The molar concentration of the buffer solution is preferably 27.5 mmol / L.

[0066] In the method provided by the present invention, the electrolyte preferably uses a HEPES buffer solution as the solvent of the reaction system. Due to the presence of the HEPES buffer solution, the pH remains stable during the entire electro-enzyme catalytic reduction reaction, and no additional compounds need to be added to adjust the pH. At the same time, the pH value of the HEPES buffer solution is 7.5, and the alkaline solution environment formed is conducive to the synthesis of NADPH, achieving green synthesis with zero carbon footprint (green power-on environment). Furthermore, the HEPES buffer solution is an environmentally friendly reagent.

[0067] In the present invention, NADP + The molar ratio of the ferredoxin-NADP+ reductase to the ferredoxin-NADP+ reductase is preferably 90000-100000:1, more preferably 90000:1. The ferredoxin-NADP+ reductase is the ferredoxin-NADP+ reductase loaded in the porous structure of the immobilized enzyme electrode.

[0068] In the present invention, the electrocatalyzed enzyme-catalyzed reduction reaction is preferably a constant voltage direct current electrocatalyzed enzyme-catalyzed reduction reaction.

[0069] In the present invention, the Ag / AgCl electrode is used as the reference electrode, and the voltage of the working electrode is preferably -0.75 to -0.8V, more preferably -0.75V.

[0070] In the present invention, the temperature of the electroenzymatic reduction reaction is preferably 5-45°C, more preferably 10-40°C; the time is preferably 4-24h, more preferably 24h; the pH value is preferably 6.8-8.2, more preferably 7.5.

[0071] The method for synthesizing NADPH by electrocatalytic immobilized enzyme provided by the present invention has a mild reaction temperature and does not require the addition of other compounds, is environmentally friendly, and the overall synthesis route achieves zero carbon footprint.

[0072] In the present invention, the electroenzyme catalytic reduction reaction is carried out under stirring conditions, and the stirring speed is 800r / min.

[0073] The specific implementation method of synthesizing NADPH using a three-electrode system of the present invention is preferably as follows: first, a reference electrode is placed in a NaCl solution, a working electrode and a counter electrode are placed in a blank HEPES buffer solution, an external power supply is turned on, a CA voltage is set to -0.75 V, a time of 86400 s, a CA graph is scanned, and after the baseline is balanced, the power supply is turned off; a NADP-containing + The buffer solution (i.e. electrolyte) makes the diluted NADP + The molar concentration of the buffer solution is 27.5mmol / L. Turn on the power again, set the CA voltage to -0.75V, and perform the electrocatalytic reduction reaction at 25℃. Monitor the current at any time during the reaction. When the current stops increasing and returns to the initial baseline, the electrocatalytic reduction reaction ends. After the reaction, take out the reaction solution, which is the NADPH generation solution.

[0074] The method provided by the present invention realizes the synthesis of NADPH by autoclave or continuous reaction, and the synthesis method is simple, easy to operate, has low production cost, short production cycle, high product purity, and more stable product. On the other hand, the present invention adopts electric-promoted immobilized enzyme to catalyze the synthesis of NADPH, and the immobilized enzyme has high efficiency, stability, and can be used repeatedly to synthesize NADPH, which reduces the production cost and cycle, and avoids the introduction of impurities generated by proteins and side reactions into the product. The synthesis method of the present invention using electric-promoted immobilized enzyme is conducive to the synthesis of high-purity and high-yield NADPH, and is conducive to a zero-carbon footprint synthesis route.

[0075] 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.

[0076] Example 1

[0077] ITO, ATO and iodine elemental solvent are dissolved in acetone to obtain a conductive filler organic solution; wherein the mass concentration of ITO is 0.9 mg / mL, the mass concentration of ATO is 0.1 mg / mL, and the concentration of iodine element is 1 mg / mL.

[0078] After ultrasonically treating the conductive filler organic solution for 30 minutes, the titanium dioxide sheet was connected to the negative pole of an external power supply and immersed in the conductive filler organic solution. The ITO glass was connected to the positive pole of an external power supply and immersed in the conductive filler organic solution. The external power supply was turned on and electrophoresis was performed at a voltage of 15V. After the treatment for 7 minutes, the external power supply was disconnected, the titanium dioxide sheet was turned over and reconnected to the external power supply, and then the external power supply was turned on and the electrophoresis was continued at a voltage of 15V for 7 minutes. A porous conductive filler coating was formed on both surfaces of the titanium dioxide sheet.

[0079] FNR was dissolved in a HEPES buffer solution (50 mmol / L, pH 7.5) to obtain a buffer solution containing FNR, which was slowly and evenly applied to the surface of the porous conductive filler coating at 4°C, wherein the mass ratio of FNR in the buffer solution containing FNR to the porous conductive filler coating was 0.017:1, and the solution was allowed to stand at 4°C for 1 hour to obtain an immobilized enzyme electrode.

[0080] NADP + Dissolved in HEPES buffer solution (50 mM, pH = 7.5) to obtain a NADP + Buffer solution (mass concentration is 0.5 g / mL), containing NADP + NADP in buffer solution + The molar ratio of FNR loaded on the immobilized enzyme electrode is 90000:1. The three-electrode system electrolytic cell was installed, the reference electrode was loaded with 0.1mM NaCl solution, the counter electrode (platinum sheet) and the immobilized enzyme electrode were loaded into HEPES buffer solution (50mM at pH = 7.5), stirring was started, the stirring speed was 800r / min, the power was turned on, the CA voltage was set to -0.75V, and the CA graph was scanned. After the baseline was balanced, NADP was added to the reactor solution. + , its molar concentration reaches 27.5mmol / L. The current is monitored at any time during the electrocatalytic enzyme reduction reaction. When the current returns to the initial baseline, the reduction reaction is completed. After the reaction is completed, the reaction solution is taken out, which is the NADPH generation solution. Figure 2 is a curve diagram showing the change of reaction current over reaction time in Example 1; Figure 3 This is an example of the purity of the reaction product prepared in Example 1. Figure 2 It can be seen that when the current value returns to the initial base value after 10 hours of reaction, the reaction is complete. Figure 3 It can be seen that the purity of NADPH in the NADPH production solution prepared in this example is 95%, and the yield is 85%.

[0081] Example 2

[0082] The immobilized enzyme electrode prepared in Example 1 was used as a working electrode to prepare NADPH according to Example 1 and was reused 5 times. The yield of the prepared NADPH in the second use was 85% and the purity was 95%; the yield of the prepared NADPH in the third use was 85% and the purity was 95%; the yield of the prepared NADPH in the fourth use was 85% and the purity was 95%; the yield of the prepared NADPH in the fifth use was 85% and the purity was 95%.

[0083] 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 synthesizing NADPH by electrocatalytic immobilized enzyme, characterized in that: The following steps are involved: A three-electrode system was used to + The buffer solution is used as the reaction solution, and the immobilized enzyme electrode is used as the working electrode to perform an electrocatalytic enzyme reduction reaction to obtain the NADPH; the reference electrode in the three-electrode system is Ag / AgCl, and the voltage of the working electrode is -0.75 to -0.8V; the counter electrode in the three-electrode system is a platinum sheet; The immobilized enzyme electrode comprises a semiconductor substrate, a porous conductive filler coating disposed on the surface of the semiconductor substrate, and ferredoxin-NADP loaded in the pore structure of the porous conductive filler coating. + Reductase; the material of the semiconductor substrate is titanium dioxide; The conductive filler is a mixture of indium tin oxide and antimony tin oxide; The preparation method of the immobilized enzyme electrode comprises the following steps: A semiconductor substrate is used as a negative electrode, an organic solution containing a conductive filler is used as an electrolyte, and a porous conductive filler coating is formed on the surface of the semiconductor substrate by an electrophoresis treatment method; Ferredoxin-NADP + The buffer solution of reductase is coated on the porous conductive filler coating, and the immobilized enzyme electrode is obtained after standing at a temperature below 25° C.; In the organic solution containing the conductive filler, the mass concentration of indium tin oxide is 0.9-1.5 mg / mL, and the mass concentration of antimony tin oxide is 0.1-0.2 mg / mL.

2. The method according to claim 1, characterized in that: The ferredoxin-NADP + The mass ratio of the reductase to the porous conductive filler coating is 0.01-0.02:

1.

3. The method according to claim 1, characterized in that: NADP + With the ferredoxin-NADP + The molar ratio of the reductase is 90,000 to 100,000:

1.

4. The method according to claim 1 or 2, characterized in that: The pore size of the porous conductive filler coating is 10-100 nm.

5. The method according to claim 1, characterized in that The temperature of the electro-enzyme catalytic reduction reaction is 5-45° C., the time is 4-24 hours, and the pH value is 6.8-8.

2.

6. The preparation method according to claim 1, characterized in that: The voltage of the electrophoresis treatment is 10-15V, and the time is 7-15min.

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