Method for catalytically synthesizing nadph using electrically promoted immobilized enzyme

Abstract

The present invention relates to the technical field of biopharmaceuticals, and in particular to a method for catalytically synthesizing NADPH using an electrically promoted immobilized enzyme. According to the present invention, a three-electrode system is used, a buffer solution containing NADP+ is used as a reaction solution, and an immobilized enzyme electrode is used as a working electrode so as to carry out an electrically-promoted enzyme catalytic reduction reaction to obtain NADPH. The immobilized enzyme electrode comprises a semiconductor substrate, a porous conductive filler coating arranged on the surface of the semiconductor substrate, and ferredoxin-NADP+reductase (FNR) loaded in a pore structure of the porous conductive filler coating. According to the present invention, the three-electrode system is used, in a power-on environment, NADP+ is used as a substrate, and the immobilized enzyme electrode loaded with FNR is used for catalytically synthesizing the NADPH. The method provided by the present invention has the characteristics of high yield, high purity, simple synthesis route, low costs and environmental friendliness.

Description

A method for synthesizing NADPH by electrocatalytic immobilized enzyme

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on June 16, 2023, with application number CN202310715036.7 and invention name “A method for electrically catalyzing the synthesis of NADPH by immobilized enzymes”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The invention belongs to the technical field of biopharmaceuticals, and particularly relates to a method for synthesizing NADPH by electrocatalytic immobilized enzyme. Background Art

[0003] Reduced Nicotinamide adenine dinucleotide phosphate (NADPH) 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.

[0004] 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 cellular protection against redox stress by providing reducing equivalents for antioxidants such as glutathione and thioredoxin. NADPH participates in the regeneration of glutathione, a key antioxidant in organisms.

[0005] Currently, NADPH is used in the biotechnology industry to produce chemicals. In chemical production, NADPH is often used to produce chiral intermediates for pharmaceuticals. 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 an NADPH-dependent ketoreductase to reduce the ketone group in the precursor compound (S)-2'-methoxy-2-methylpropiophenone. This ketoreductase catalyzes the stereospecific reduction of the ketone group to produce (S)-2'-methoxy-2-methylpropionic acid methyl ester with high enantioselectivity. This intermediate is then converted to (S)-naproxen through a series of chemical reactions.

[0006] 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.

[0007] NADPH is NADP + The reduced form of NADPH. Currently, the methods for synthesizing NADPH can be divided into bio-fermentation and chemical synthesis. The bio-fermentation method is to separate and purify various products of microorganisms using bio-fermentation technology or other microbial culture technologies. The bio-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 the disadvantages of many reaction steps, harsh reaction conditions, the need for 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.

[0008] The enzyme-catalyzed synthesis of NADPH is a highly efficient and selective synthesis reaction route with a product that has specific selectivity, mild reaction conditions, high yield, and friendly reaction reagent conditions. Currently, the most commonly used enzymes for the production of NADPH are formate dehydrogenase (GDH) and glucose dehydrogenase (FDH). GDH is highly active, but requires additional glucose as a substrate, and the byproduct gluconic acid easily leads to enzyme inactivation, resulting in a decrease in yield, and subsequent separation and purification of NADPH also faces huge challenges. On the other hand, FDH facilitates the subsequent separation and purification of the product NADPH, but produces carbon dioxide as a byproduct during the production process, which pollutes the environment.

[0009] In summary, the current enzymatic production of NADPH has the problems of low yield and serious pollution.

[0010] Summary of the Invention

[0011] The purpose 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.

[0012] In order to achieve the above object, the present invention provides the following technical solutions:

[0013] The present invention provides a method for synthesizing NADPH by electrocatalytic immobilized enzyme, comprising the following steps:

[0014] 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 electroenzyme-catalyzed reduction reaction to obtain the NADPH;

[0015] 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.

[0016] Preferably, the mass ratio of the ferredoxin-NADP+ reductase to the porous conductive filler coating is 0.01-0.02:1.

[0017] Preferably, NADP + The molar ratio of the precipitate to the ferredoxin-NADP+ reductase is 90,000 to 100,000:1.

[0018] 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.

[0019] Preferably, the semiconductor substrate is made of titanium dioxide.

[0020] 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.

[0021] 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.

[0022] Preferably, the preparation method of the immobilized enzyme electrode comprises the following steps:

[0023] 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;

[0024] A buffer solution containing ferredoxin-NADP+ reductase is coated on the porous conductive filler coating, and the solution is allowed to stand at a temperature below 25° C. to obtain the immobilized enzyme electrode.

[0025] Preferably, 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 0.9-1.5 mg / mL, and the mass concentration of antimony tin oxide is 0.1-0.2 mg / mL.

[0026] Preferably, the voltage of the electrophoresis treatment is 10-15 V, and the time is 7-15 min.

[0027] 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 provided 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, and in an electric environment, NADP + As a substrate, NADPH is synthesized by an immobilized enzyme electrode loaded with ferredoxin-NADP+ reductase (FNR). The method provided by the present invention has the following advantages:

[0028] The method for synthesizing NADPH by electrocatalytic immobilized enzyme provided by the present invention can realize continuous reaction synthesis of NADPH under an electric environment, shorten the production cycle (reaction time is controlled within 24 hours), and improve the purity and yield of NADPH (purity ≥95%, yield ≥85%).

[0029] The present invention uses an immobilized enzyme electrode to synthesize NADPH. The immobilized enzyme electrode can be used repeatedly, thereby improving the utilization efficiency of FNR and reducing production costs. On the other hand, the present invention uses an immobilized enzyme electrode to produce NADPH, which makes NADPH easy to separate, avoids protein residue, simplifies the purification steps, and improves product purity.

[0030] 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.

[0031] The method for synthesizing NADPH by electrocatalysis using immobilized enzymes provided by the present invention does not require the addition of other compounds, thus avoiding the introduction of impurities and facilitating the improvement of the purity of NADPH. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 is a schematic diagram of the NADPH synthesis principle provided by an embodiment of the present invention;

[0033] FIG2 is a graph showing the reaction current versus reaction time in Example 1;

[0034] FIG3 is an example diagram showing the purity of the reaction product prepared in Example 1. DETAILED DESCRIPTION

[0035] The present invention provides a method for synthesizing NADPH by electrocatalytic immobilized enzyme, comprising the following steps:

[0036] 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 electroenzyme-catalyzed reduction reaction to obtain the NADPH;

[0037] 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.

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

[0039] The method provided by the present invention adopts a three-electrode system, which includes a working electrode, a reference electrode and a counter electrode.

[0040] In the present invention, the working electrode is an immobilized enzyme electrode.

[0041] 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.

[0042] The immobilized enzyme electrode provided by the present invention includes 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). 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.

[0043] 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.

[0044] 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 between the coating and 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.

[0045] In the present invention, the porous conductive filler coating is preferably provided on both surfaces of the semiconductor substrate and has a thickness of preferably 3 to 10 mm.

[0046] 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.

[0047] In the present invention, the mass ratio of the ferredoxin-NADP+ reductase to the porous conductive filler coating is preferably 0.01 to 0.02:1, more preferably 0.017:1.

[0048] In the present invention, the method for preparing the immobilized enzyme electrode preferably comprises the following steps:

[0049] 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;

[0050] A buffer solution containing ferredoxin-NADP+ reductase is coated on the porous conductive filler coating, and the immobilized enzyme electrode is obtained after standing.

[0051] 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.

[0052] In the present invention, the organic solution containing the conductive filler preferably comprises the conductive filler, an organic solvent, and elemental iodine. The organic solvent is preferably acetone. The elemental iodine is used to improve the conductivity of the organic solution containing the conductive filler.

[0053] 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 to 1.5 mg / mL, more preferably 0.9 mg / mL; the mass concentration of antimony tin oxide is preferably 0.1 to 0.2 mg / mL, more preferably 0.1 mg / mL. In the present invention, the mass concentration of elemental iodine in the organic solution containing the conductive filler is preferably 1 mg / mL.

[0054] 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.

[0055] 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 to 500W.

[0056] 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 minutes.

[0057] In the present invention, the specific implementation method of the electrophoretic treatment is preferably: connecting the semiconductor substrate to the negative pole of an external power source and immersing it in an organic solution containing a conductive filler; connecting the ITO glass to the positive pole of an external power source and immersing it in an organic solution containing a conductive filler; turning 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 to prepare a porous conductive filler coating on both surfaces of the semiconductor substrate; the electrophoretic treatment time for each surface of the semiconductor substrate is preferably 7 minutes. In a specific embodiment of the present invention, the present invention preferably performs the electrophoretic treatment on the external power source for 7 minutes, then turns the semiconductor substrate over and connects the external power source to continue the electrophoretic treatment on the other surface of the semiconductor substrate.

[0058] 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 allows it to stand at a temperature below 25° C. to obtain the immobilized enzyme electrode.

[0059] In the present invention, the buffer solution containing ferredoxin-NADP+ reductase preferably comprises ferredoxin-NADP+ reductase and a buffer solution, and the buffer solution is preferably a HEPES buffer solution. The HEPES buffer solution preferably has a molar concentration of 50 mmol / L and a pH of 7.5. The pH of the buffer solution containing ferredoxin-NADP+ reductase is preferably 7.5.

[0060] The present invention preferably coats a buffer solution containing ferredoxin-NADP+ reductase on the porous conductive coatings disposed on both surfaces of the semiconductor substrate. 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.

[0061] In the present invention, the standing time is preferably 1 hour.

[0062] In the present invention, the reference electrode is preferably an Ag / AgCl electrode.

[0063] In the present invention, the counter electrode is preferably a platinum sheet.

[0064] In the present invention, the reference electrode is placed in a NaCl solution, and the molar concentration of the NaCl solution is preferably 0.1 mmol / L.

[0065] In the present invention, the working electrode and the counter electrode are disposed in an electrolyte.

[0066] In the present invention, the reaction solution contains NADP + of buffer solution.

[0067] In the present invention, NADP is contained + The buffer solution preferably includes NADP + and HEPES buffer solution. The molar concentration of the HEPES buffer solution is preferably 50 mmol / L. Containing NADP + The pH value of the buffer solution is preferably 7.5. + The molar concentration of the buffer solution is preferably 27.5 mmol / L.

[0068] 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 throughout the electroenzymatic reduction reaction, eliminating the need for the addition of other compounds to adjust the pH. Furthermore, the pH value of the HEPES buffer solution is 7.5, creating an alkaline environment that is conducive to the synthesis of NADPH, achieving a zero-carbon-footprint green synthesis (green power-on environment). Furthermore, the HEPES buffer solution is an environmentally friendly reagent.

[0069] In the present invention, NADP + The molar ratio of the ferredoxin-NADP+ reductase to the ferredoxin-NADP+ reductase is preferably 90,000 to 100,000: 1, more preferably 90,000: 1. The ferredoxin-NADP+ reductase is the ferredoxin-NADP+ reductase loaded in the porous structure of the immobilized enzyme electrode.

[0070] In the present invention, the electrocatalytic enzyme reduction reaction is preferably a constant voltage direct current electrocatalytic enzyme reduction reaction.

[0071] 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 -0.8 V, more preferably -0.75 V.

[0072] 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; and the pH value is preferably 6.8-8.2, more preferably 7.5.

[0073] 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 a zero carbon footprint.

[0074] In the present invention, the electroenzyme-catalyzed reduction reaction is carried out under stirring conditions, and the stirring speed is 800 r / min.

[0075] The preferred embodiment of the present invention using a three-electrode system to synthesize NADPH is as follows: first, the reference electrode is placed in a NaCl solution, the working electrode and the counter electrode are placed in a blank HEPES buffer solution, the external power supply is turned on, the CA voltage is set to -0.75V, the time is 86400s, and the CA graph is scanned. After the baseline is balanced, the power supply is turned off; NADP is added to the HEPES buffer solution. + Buffer solution (ie electrolyte) so that the diluted NADP + The molar concentration of the buffer solution was 27.5 mmol / L. The power was turned on again, the CA voltage was set to -0.75 V, and the electrocatalytic reduction reaction was carried out at 25°C. The current was monitored at all times during the reaction. The electrocatalytic reduction reaction was terminated when the current stopped increasing and returned to the initial baseline. After the reaction was completed, the reaction solution was removed, which was the NADPH production solution.

[0076] The method provided by the present invention realizes the synthesis of NADPH by kettle or continuous reaction. 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. The immobilized enzyme has high efficiency, stability and repeated use to synthesize NADPH, which reduces 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.

[0077] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0078] Example 1

[0079] ITO, ATO and iodine were dissolved in acetone to obtain a conductive filler organic solution; wherein the mass concentration of ITO was 0.9 mg / mL, the mass concentration of ATO was 0.1 mg / mL, and the concentration of iodine was 1 mg / mL.

[0080] 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 treatment was performed at a voltage of 15V. After 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 electrophoresis treatment 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.

[0081] 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. The mass ratio of FNR in the FNR-containing buffer solution to the porous conductive filler coating was 0.017:1. The solution was allowed to stand at 4°C for 1 hour to obtain an immobilized enzyme electrode.

[0082] NADP + Dissolved in HEPES buffer solution (50 mM, pH = 7.5) to obtain a solution containing NADP + Buffer solution (mass concentration is 0.5g / mL), which contains NADP + NADP in buffer solution + The molar ratio of FNR loaded on the immobilized enzyme electrode was 90000:1. The three-electrode 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 at 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, and the current is monitored at any time during the electroenzyme-catalyzed reduction reaction. When the current returns to the initial baseline, the reduction reaction is terminated. After the reaction is completed, the reaction solution is taken out, which is the generated solution of NADPH. Figure 2 is a curve diagram of the change of reaction current with reaction time in Example 1; Figure 3 is an example diagram of the purity of the reaction product prepared in Example 1. It can be seen from Figure 2 that when the current value returns to the initial base value after 10 hours of reaction of the present invention, the reaction is terminated. It can be seen from Figure 3 that the purity of NADPH in the generated solution of NADPH prepared in this example is 95%, and the yield is 85%.

[0083] Example 2

[0084] The immobilized enzyme electrode prepared in Example 1 was used as a working electrode to prepare NADPH according to Example 1 and 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%; and the yield of the prepared NADPH in the fifth use was 85% and the purity was 95%.

[0085] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for synthesizing NADPH by electrocatalytic immobilized enzyme. It is 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 electro-enzyme catalytic reduction reaction to obtain the NADPH; 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.

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

1.

3. The method according to claim 1 or 2, It is characterized in that NADP in the reaction solution + The molar ratio of the precipitate to the ferredoxin-NADP+ reductase is 90,000 to 100,000:

1.

4. The method according to claim 1 or 2, It is characterized in that 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 10-100 nm.

5. The method according to claim 4, It is characterized in that In the mixture of indium tin oxide and antimony tin oxide, the mass ratio of ITO to ATO is (0.9-1.5):(0.1-0.2).

6. The method according to claim 1 or 2, It is characterized in that The thickness of the porous conductive filler coating is 3 to 10 mm.

7. The method according to claim 1, It is characterized in that The semiconductor substrate is made of titanium dioxide.

8. The method according to claim 1, It is characterized in that The reference electrode in the three-electrode system is Ag / AgCl, and the voltage of the working electrode is -0.75 to -0.8V.

9. The method according to claim 1, It is 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.

10. The method according to claim 1, It is characterized in that 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; 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.

11. The method according to claim 10, It is characterized in that The organic solution containing the conductive filler includes the conductive filler, an organic solvent and iodine element.

12. The method according to claim 11, It is characterized in that In the organic solution containing the conductive filler, the mass concentration of iodine element is preferably 1 mg / mL.

13. The method according to claim 10 or 11, It is characterized in that 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.

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

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