A hollow ZIF-8 immobilized dual enzyme and its preparation method and application

Abstract

The present invention discloses a hollow ZIF-8 immobilized bienzyme and its preparation method and application, by embedding formate dehydrogenase and carbonic anhydrase in ZIF-67, then growing a layer of ZIF-8 shell on its surface, dissociating ZIF-67, and the formate dehydrogenase and carbonic anhydrase are fixed in the ZIF-8 shell in a free state, thereby maintaining a high enzyme activity to form a hollow ZIF-8 immobilized bienzyme. Utilizing 2-hydroxy-1,4-naphthoquinone coenzyme regeneration, bioelectrocatalytic CO2 reduction is achieved at the cathode. The preparation process of this method is simple to operate and easy to implement. The hollow structure is used to provide a soft microenvironment for the enzyme. Hollow ZIF-8 and carbonic anhydrase can increase the substrate CO2 adsorption capacity. Using 2-hydroxy-1,4-naphthoquinone regeneration can reduce the consumption of expensive coenzymes, and bioelectrocatalytic CO2 reduction has a higher formic acid yield.

Description

Technical Field

[0001] The present invention relates to a method and application of using a multifunctional carrier of immobilized enzyme to enhance enzyme electrocatalytic CO2 reduction, and specifically to the preparation of hollow carrier, immobilized enzyme and bioelectrocatalytic coenzyme regeneration application. Background Art

[0002] Global warming, caused by emissions of carbon dioxide, nitrous oxide, methane, and hydrofluorocarbons, poses a serious environmental threat. Excessive carbon dioxide emissions, as a greenhouse gas, undoubtedly contribute to the global warming effect, leading to more frequent extreme weather events, desertification, and sea level rise. Therefore, capturing and fully utilizing carbon dioxide to convert it into high-value-added carbon-containing chemicals and energy compounds is of great value.

[0003] Among the numerous CO2 utilization pathways, enzyme catalysis provides a promising alternative due to its superior selectivity. Formate is the first stable intermediate in the reduction of CO2 through a cascade reaction, and reducing CO2 to formate is the most economical route. Formate dehydrogenase (FDH) is an oxidoreductase that specifically and reversibly catalyzes the conversion of CO2 to formate. In this process, reduced nicotinamide adenine dinucleotide (NADH) is usually required as a sacrificial agent to shuttle electrons and protons between FDH and CO2, accelerating the reversible reduction of CO2. However, due to its high price and NAD + The large-scale use of enzymatic reduction of CO2 is limited by the inhibition of product formation. At atmospheric pressure, the solubility of CO2 in water is limited, which may lead to low substrate concentration. Therefore, increasing the solubility of CO2 is crucial to promoting the forward reaction. The zeolite imidazole framework ZIF-8 is considered to be an excellent porous material for selective capture of CO2 with a very high CO2 adsorption capacity. In addition, carbonic anhydrase (CA) plays a vital role in the capture and conversion of CO2 in organisms. CA can accelerate the absorption of CO2 in aqueous solution and only requires low desorption heat and energy. However, free FDH and CA have problems such as easy deactivation, difficult recovery, and high cost when catalyzing the reaction.

[0004] Therefore, in order to achieve the reuse of enzymes, ensure the efficient reduction efficiency of CO2, and reduce the consumption of expensive coenzymes, it is urgent to develop a carrier that can effectively adsorb CO2, immobilize enzymes and realize coenzyme regeneration. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention proposes a hollow ZIF-8 immobilized dual enzyme and its preparation method, which utilizes MOFs to immobilize formate dehydrogenase and carbonic anhydrase for enzymatic electrocatalytic reduction of CO2, thereby achieving efficient and low-cost CO2 conversion. The preparation method of the present invention is simple to prepare and easy to implement. By embedding formate dehydrogenase and carbonic anhydrase in ZIF-67, a layer of ZIF-8 shell is then grown on its surface to dissociate the ZIF-67, allowing FDH and CA to be fixed in a free state in the ZIF-8 cavity for enzymatic electrocatalytic CO2 reduction. 2-Hydroxy-1,4-naphthoquinone is used to regenerate coenzymes, reducing the consumption of expensive coenzymes. The bioelectrocatalytic CO2 reduction has a high formic acid yield.

[0006] This invention utilizes MOFs to encapsulate the enzyme within a cavity. The hollow structure reduces interfacial interactions, resulting in fewer structural constraints and minimal forces between the enzyme and the support, maximizing molecular freedom and maintaining high enzyme activity. The MOF protects the free enzyme from macromolecular inhibitors. By utilizing carbonic anhydrase to adsorb CO₂ and selecting a support with a high CO₂ adsorption capacity for the immobilized enzyme, the substrate concentration is increased.

[0007] To solve the above technical problems, the present invention proposes a hollow ZIF-8 immobilized dual enzyme, which includes formate dehydrogenase and carbonic anhydrase. The dual enzyme is embedded in ZIF-67, and then a layer of ZIF-8 shell is grown on its surface. The ZIF-67 is dissociated, and the formate dehydrogenase and carbonic anhydrase are immobilized in the ZIF-8 shell in a free state to form a hollow ZIF-8 immobilized dual enzyme. The preparation steps are as follows:

[0008] Step 1) adding an aqueous solution of hexadecyltrimethylammonium bromide to an aqueous solution of 2-methylimidazole to obtain a mixed solution A, stirring uniformly, adding formate dehydrogenase and carbonic anhydrase, and then adding an aqueous solution of cobalt nitrate hexahydrate, stirring uniformly at room temperature to form a reaction system A, standing at 4°C for 1 hour, and centrifuging to separate a precipitate, which is recorded as FDH&CA@ZIF-67 precipitate;

[0009] Step 2) adding an aqueous solution of hexadecyltrimethylammonium bromide to an aqueous solution of 2-methylimidazole to obtain a mixed solution B, wherein the concentration and amount of the aqueous solution of 2-methylimidazole are consistent with those in reaction system A; after stirring evenly, dispersing the FDH&CA@ZIF-67 precipitate obtained in step 1) into the mixed solution B, then adding an aqueous solution of zinc nitrate hexahydrate, stirring evenly at room temperature to form a reaction system B, standing at 4°C for 1 hour, collecting the precipitate by filtration, washing the precipitate with excess deionized water multiple times, and freeze-drying for more than 12 hours. The obtained product is recorded as FDH&CA@ZIF-67@ZIF-8 precipitate;

[0010] Step 3) The product obtained in step 2) is immersed in deionized water and incubated at room temperature for 24 hours. The precipitate is collected by filtration, washed with excess deionized water multiple times, and freeze-dried for more than 12 hours. The obtained product is a hollow ZIF-8 immobilized dual enzyme.

[0011] Furthermore, the preparation method of the present invention, wherein:

[0012] In the mixed solution A and the mixed solution B, the concentration of the cetyltrimethylammonium bromide aqueous solution is 1.925 mmol / L, and the concentration of the 2-methylimidazole aqueous solution is 1.843 M; in the mixed solution A, the volume ratio of the cetyltrimethylammonium bromide aqueous solution to the 2-methylimidazole aqueous solution is 2:3; in the mixed solution B, the volume ratio of the cetyltrimethylammonium bromide aqueous solution to the 2-methylimidazole aqueous solution is 4:3.

[0013] In the reaction system A, the mass volume ratio of the formate dehydrogenase to all aqueous solutions in the reaction system A is 1 to 2.5 mg / mL, and the mass ratio of the carbonic anhydrase to formate dehydrogenase is 1:0.5 to 3; the mass volume ratio of the formate dehydrogenase to all aqueous solutions in the reaction system A is 2 mg / mL, and the mass ratio of the carbonic anhydrase to formate dehydrogenase is 1:2; the concentration of the added cobalt nitrate hexahydrate aqueous solution is 97.5 mmol / L, and the volume ratio of the cobalt nitrate hexahydrate aqueous solution to the 2-methylimidazole aqueous solution is 1:3.

[0014] In the reaction system B, the concentration of the added zinc nitrate hexahydrate aqueous solution was 97.5 mmol / L, and the volume ratio of the zinc nitrate hexahydrate aqueous solution to the 2-methylimidazole aqueous solution was 1:3.

[0015] The hollow ZIF-8 immobilized dual enzyme prepared by the present invention is used for bioelectrocatalytic reduction of CO2 to formic acid, and the process is as follows:

[0016] An appropriate amount of deionized water was added to the anode chamber, and a certain amount of hollow ZIF-8 immobilized dual enzyme, 2-hydroxy-1,4-naphthoquinone and reduced coenzyme I was added to the cathode chamber, wherein the mass volume ratio of the hollow ZIF-8 immobilized dual enzyme was 5-9 mg / mL, the molar concentration of 2-hydroxy-1,4-naphthoquinone was 2.5 mmol / L, the molar concentration of reduced coenzyme I was 3.5 mmol / L, and the molar ratio of 2-hydroxy-1,4-naphthoquinone to reduced coenzyme I was 2.5:3.5; Tris-HCl buffer with a concentration of 50 mmol / L and pH = 6.5 was used as the reaction solvent, CO2 was bubbled at a flow rate of 100 mL / min, the applied voltage was -0.65 V, and the reference electrode was Ag / AgCl, thereby realizing bioelectrocatalytic CO2 reduction, electrolyzing water in the anode chamber to generate protons and electrons, the protons entered the cathode chamber through the proton exchange membrane, and the electrons were captured by 2-hydroxy-1,4-naphthoquinone and NAD + The reaction was carried out to achieve continuous regeneration of reduced coenzyme I; the concentration of formic acid produced after 180 minutes was 1.11-3.12 mmol / L.

[0017] The hollow ZIF-8 immobilized dual enzyme prepared by the present invention has good operational stability as a catalyst for bioelectrocatalytic CO2 reduction. After 8 cycles of reaction, the relative activity retention of the hollow ZIF-8 immobilized dual enzyme can be as high as 80.1%.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The present invention prepares a hollow MOF-embedded enzyme. The hollow structure reduces the interfacial interaction between the enzyme and the MOF, and its structure is less restricted, and the interaction force between the enzyme and the carrier is small, which maximizes the freedom of the molecule and maintains a high enzyme activity.

[0020] (2) The preparation method of the present invention embeds FDH and CA so that they exist in a free state in the carrier cavity, utilizes the high capture ability of the carrier and carbonic anhydrase for CO2, thereby increasing the local concentration of CO2 and promoting the CO2 reduction reaction.

[0021] (3) The present invention uses 2-hydroxy-1,4-naphthoquinone as an electron mediator to achieve coenzyme regeneration, thereby reducing the consumption of expensive coenzymes.

[0022] (4) The materials and reagents used for the immobilized enzyme and coenzyme regeneration of the present invention are conventional mass-produced products with low prices.

[0023] (5) The method of the present invention is used for resource processing of CO2, specifically for bioelectrocatalytic reduction of CO2 into high value-added product formic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1is the XRD pattern of FDH&CA@ZIF-67 obtained in Example 1;

[0025] Figure 2 is the XRD pattern of ZIF-8, ZIF-67 and FDH&CA@ZIF-67@ZIF-8 obtained in Example 1;

[0026] Figure 3 are the transmission and scanning images of FDH&CA@ZIF-67@ZIF-8 obtained in Example 1;

[0027] Figure 4 is the XRD pattern of FDH&CA@H-ZIF-8 prepared in Example 1;

[0028] Figure 5 This is a transmission electron microscope image of FDH&CA@H-ZIF-8 prepared in Example 1;

[0029] Figure 6 This is a laser confocal image of FDH&CA@H-ZIF-8 prepared in Example 1;

[0030] Figure 7 is a diagram of optimizing the CA and FDH concentration conditions in Example 2;

[0031] Figure 8 This is a schematic diagram of the electrocatalytic CO2 reduction of the dual-enzyme microreactor constructed in Example 3;

[0032] Figure 9 This is a graph showing the effect of repeated use of FDH&CA@H-ZIF-8 under the conditions of Example 3;

[0033] Figure 10 Time-dependent changes in formic acid production in various enzymatic reaction systems at an applied voltage of -0.65 V. Solid symbols indicate NADH regeneration; hollow symbols indicate no NADH regeneration. DETAILED DESCRIPTION

[0034] The design concept of the present invention is to propose a method of utilizing the hollow structure of the carrier to protect the enzyme and the high capture ability of MOF and carbonic anhydrase for CO2, to construct a hollow MOF immobilized enzyme (FDH&CA@H-ZIF-8), and then combine it with electrocatalytic NADH regeneration to enable CO2 to be efficiently reduced to formic acid in the enzyme electrocatalytic system. By pre-encapsulating FDH and CA in ZIF-67, and then growing a layer of ZIF-8 shell on its surface, ZIF-67 is dissociated to form a ZIF-8 cavity. The enzyme originally fixed in ZIF-67 will be released and encapsulated in the cavity between the ZIF-8 shells. The addition of CA greatly promotes the capture of CO2, significantly improving the CO2 capture ability of FDH&CA@H-ZIF-8, thereby accelerating the further conversion to formic acid under FDH catalysis.

[0035] The present invention will be described below with reference to the accompanying drawings and specific embodiments to provide a further understanding of the present invention. The following embodiments are by no means intended to limit the present invention in any way.

[0036] Example 1: Preparation of hollow ZIF-8 immobilized dual enzyme, the preparation process is as follows:

[0037] 1) FDH and CA were embedded in ZIF-67 by embedding method. The obtained product was recorded as FDH&CA@ZIF-67. The specific process was as follows:

[0038] 5 mL of a 1.925 mmol / L aqueous solution of hexadecyltrimethylammonium bromide (CTAB) was added to 7.5 mL of a 1.843 M aqueous solution of 2-methylimidazole, and the mixture was stirred at 500 rpm for 5 minutes. An aqueous solution containing 30 mg of FDH and 15 mg of CA was then added to the mixture, followed by 2.5 mL of a 97.5 mmol / L aqueous solution of Co(NO₃)₂ 6H₂O. The resulting mixture was stirred at room temperature for 5 minutes. The resulting reaction solution was allowed to stand in a refrigerator at 4°C for 1 hour and centrifuged to obtain a precipitate of FDH and CA@ZIF-67.

[0039] The prepared FDH&CA@ZIF-67 was characterized by XRD. Figure 1 As shown, crystal peaks appeared at 2θ=7.2, 10.2, 12.6, 14.7, 16.5 and 17.9, proving the successful preparation of ZIF-67. The fixation of FDH and CA had no effect on the crystal structure of ZIF-67.

[0040] 2) A ZIF-8 shell was grown on the surface of FDH&CA@ZIF-67. The resulting product was denoted as FDH&CA@ZIF-67@ZIF-8. The specific process was as follows:

[0041] First, the FDH&CA@ZIF-67 obtained in step 1) was dispersed in a mixture of 7.5 mL of 1.843 M 2-methylimidazole aqueous solution and 10 mL of 1.925 mmol / L CTAB aqueous solution. Then, 2.5 mL of 97.5 mmol / L Zn(NO₃)₂ 6H₂O aqueous solution was added, and the resulting mixture was stirred at 500 rpm for 5 minutes. The resulting reaction solution was allowed to stand in a refrigerator at 4°C for 1 hour. The FDH&CA@ZIF-67@ZIF-8 precipitate was collected by filtration and washed multiple times with excess deionized water. The resulting product, FDH&CA@ZIF-67@ZIF-8, was freeze-dried overnight.

[0042] The prepared FDH&CA@ZIF-67@ZIF-8 was characterized and the results were as follows Figure 2 As shown in the figure, crystal peaks appeared at 2θ=7.2, 10.2, 12.6, 14.7, 16.5 and 17.9, and after a layer of ZIF-8 was transiently grown outside ZIF-67, the strong crystallinity of ZIF-8 was revealed. Figure 3 It can be clearly seen that the MOF exhibits a uniform solid structure after the enzyme is immobilized.

[0043] 3) Hollowing FDH&CA@ZIF-67@ZIF-8 was performed through a gentle hollowing process, and the obtained product was recorded as FDH&CA@H-ZIF-8. The specific process is as follows:

[0044] The FDH&CA@ZIF-67@ZIF-8 precipitate obtained in step 2 was incubated with 200 mL of deionized water at room temperature for 24 h for hollowing. After this process, the FDH&CA@H-ZIF-8 precipitate was collected by filtration and washed multiple times with excess deionized water. The resulting product was freeze-dried overnight to obtain a hollow ZIF-8 immobilized dual enzyme, which was recorded as FDH&CA@H-ZIF-8.

[0045] The prepared FDH&CA@H-ZIF-8 was characterized and the results showed Figure 4 As shown, FDH&CA@H-ZIF-8 showed crystal peaks at 2θ=7.2, 10.2, 12.6, 14.7, 16.5 and 17.9, proving the successful preparation of FDH&CA@H-ZIF-8. The hollowing did not cause changes in the crystal, maintaining the rigid structure of ZIF-8, and had certain advantages. Figure 5 TEM results show that after the hollowing process, the MOF crystals formed a central cavity without changing their morphology.

[0046] CA and FDH were fluorescently labeled with Rhodamine B and FITC, respectively. Figure 6 As shown in a and b, fluorescently labeled CA and FDH emit red and green fluorescence respectively after being excited by a certain wavelength. Figure 6 Figure c shows that the two enzymes are co-encapsulated and emit yellow fluorescence, confirming the presence of dual enzymes in FDH&CA@H-ZIF-8.

[0047] Example 2: Optimizing the concentrations of CA and FDH in the preparation process of the present invention

[0048] Six different hollow ZIF-8 immobilized dual enzyme samples were prepared according to the preparation process of Example 1. The amounts and concentrations of CA and FDH in step 1) are shown in Table 1.

[0049] Table 1

[0050]

[0051] The enzyme-catalyzed CO2 reduction reaction with electrocatalytic NADH regeneration was carried out in a three-electrode system consisting of a cathode chamber, a proton exchange membrane, and an anode chamber. 10 mL of deionized water was added to the anode chamber, and 50 mg of the sample prepared in Example 2 and an appropriate amount of 2-hydroxy-1,4-naphthoquinone (HNQ) and reduced coenzyme I (NADH) were added to the cathode chamber. The concentration of HNQ in each experiment was 2.5 mmol / L, and the concentration of NADH was 3.5 mmol / L. The six samples were subjected to the experiment of bioelectrocatalytic CO2 reduction to formic acid; 10 mL of Tris (50 mmol / L, pH = 6.5) was used as the reaction solvent. At the same time, CO2 was bubbled (100 mL / min) and the applied voltage was -0.65 V (vs. Ag / AgCl), thereby achieving bioelectrocatalytic CO2 reduction. From Table 1 and Figure 7 As can be seen, at the same FDH concentration of 1 mg / mL, the reaction rate increases with increasing CA concentration, demonstrating that CA can promote CO2 conversion by accelerating the hydration of carbon dioxide. However, when the FDH concentration increases to 2.5 mg / mL, increasing CA concentration does not effectively accelerate the overall reaction; the reaction rate at this point is the same as when the FDH concentration is 2 mg / mL. Furthermore, from a reaction kinetics perspective, increasing the FDH content is more conducive to the forward direction of the reaction. The best results were achieved when CA and FDH concentrations were 1 mg / mL and 2 mg / mL, respectively, resulting in the greatest amount of formic acid.

[0052] Example 3: A dual-enzyme microreactor was constructed using the FDH & CA@H-ZIF-8 prepared in Example 1 for bioelectrochemical coupled CO2 reduction. The specific operation process is as follows:

[0053] The enzymatic CO2 reduction reaction with electrocatalytic NADH regeneration was carried out in a three-electrode system consisting of a cathode chamber, a proton exchange membrane, and an anode chamber. 10 mL of deionized water was added to the anode chamber, and 90 mg of FDH&CA@H-ZIF-8 obtained in Example 1, HNQ (2 mmol / L), and NADH (3.5 mmol / L) were added to the cathode chamber. 10 mL of Tris (50 mmol / L, pH = 6.5) was used as the reaction solvent. CO2 was bubbled (100 mL / min) and an applied voltage of -0.65 V (vs. Ag / AgCl) was applied to achieve bioelectrocatalytic CO2 reduction.

[0054] Figure 8 Schematic diagram of FDH&CA@H-ZIF-8 enzyme electrocatalytic CO2 reduction. In the cathode chamber, FDH uses NADH as a reducing agent to reduce CO2. Water is electrolyzed in the anode chamber to generate protons and electrons. Protons enter the cathode chamber through the proton exchange membrane, and electrons are captured by HNQ and NAD + reaction to achieve continuous regeneration of NADH.

[0055] Electrochemically coupled enzyme-catalyzed CO2 reduction was performed using the method in Example 3. The concentration of formic acid produced after 180 minutes was 3.12 mmol / L.

[0056] Example 4: According to the reaction conditions of Example 3, the prepared FDH&CA@H-ZIF-8 was subjected to 8 cycles of reaction, and the activity of formate dehydrogenase after each reaction was measured to evaluate its performance. Figure 9 It can be seen that 80.1% of the enzyme activity can still be retained after being reused 8 times, indicating that the catalyst has good operational stability.

[0057] Comparative Example: Ten enzymatic reaction systems were designed to compare and investigate the performance of FDH&CA@H-ZIF-8 under actual reaction conditions. Figure 10As shown, the free enzyme produced a maximum of 0.425 mmol / L of formic acid within 3 hours. After FDH was encapsulated in ZIF-67@ZIF-8 and coupled with electrochemical NADH regeneration, the FDH@ZIF-67@ZIF-8 system produced a maximum of 1.41 mmol / L of formic acid within 3 hours. When FDH was encapsulated in a hollow ZIF-8 system coupled with electrochemical regeneration (FDH@H-ZIF-8), the formic acid yield reached 1.92 mmol / L. Furthermore, after co-encapsulation of CA, the resulting FDH&CA@H-ZIF-8 produced 3.12 mmol / L of formate within 3 hours in an electrochemical NADH regeneration system. In summary, FDH&CA@H-ZIF-8 exhibited the highest formic acid yield in the enzymatic electrocatalytic CO2 reduction system, at 3.12 mmol / L, which is 7.3 times that of the free enzyme system.

[0058] The dual-enzyme microreactor obtained by the preparation method of the invention can enhance the adsorption of CO2, which can be used for the resource treatment of CO2 to produce formic acid, a chemical with high added value, and has a good formic acid production and good usability.

[0059] Although the present invention has been described above in conjunction with the accompanying drawings, the present invention is not limited to the above-mentioned specific embodiments. The above-mentioned specific embodiments are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can make many improvements and changes without departing from the purpose of the present invention, which are all protected by the present invention.

Claims

1. A method for preparing a hollow ZIF-8 immobilized dual enzyme, characterized in that: The dual enzyme comprises formate dehydrogenase and carbonic anhydrase, and is prepared by embedding the formate dehydrogenase and carbonic anhydrase in ZIF-67, then growing a layer of ZIF-8 shell on the surface of the ZIF-67, and dissociating the ZIF-67. The formate dehydrogenase and carbonic anhydrase are immobilized in the ZIF-8 shell in a free state to form a hollow ZIF-8 immobilized dual enzyme; the preparation method comprises the following steps: Step 1) adding an aqueous solution of hexadecyltrimethylammonium bromide to an aqueous solution of 2-methylimidazole to obtain a mixed solution A, stirring uniformly, adding formate dehydrogenase and carbonic anhydrase, and then adding an aqueous solution of cobalt nitrate hexahydrate, stirring uniformly at room temperature to form a reaction system A, standing at 4°C for 1 hour, and centrifuging to separate a precipitate, which is recorded as FDH&CA@ZIF-67 precipitate; Step 2) adding an aqueous solution of hexadecyltrimethylammonium bromide to an aqueous solution of 2-methylimidazole to obtain a mixed solution B, wherein the concentration and amount of the aqueous solution of 2-methylimidazole are consistent with those in reaction system A; after stirring evenly, dispersing the FDH&CA@ZIF-67 precipitate obtained in step 1) into the mixed solution B, then adding an aqueous solution of zinc nitrate hexahydrate, stirring evenly at room temperature to form a reaction system B, standing at 4°C for 1 hour, collecting the precipitate by filtration, washing the precipitate with excess deionized water multiple times, and freeze-drying for more than 12 hours. The obtained product is recorded as FDH&CA@ZIF-67@ZIF-8 precipitate; Step 3) immersing the product obtained in step 2) in deionized water, incubating at room temperature for 24 hours, collecting the precipitate by filtration, washing the precipitate with excess deionized water multiple times, and freeze-drying for more than 12 hours to obtain a hollow ZIF-8 immobilized dual enzyme; In the mixed solution A and the mixed solution B, the concentration of the cetyltrimethylammonium bromide aqueous solution was 1.925 mmol / L, and the concentration of the 2-methylimidazole aqueous solution was 1.843 M. In the mixed solution A, the volume ratio of the cetyltrimethylammonium bromide aqueous solution to the 2-methylimidazole aqueous solution was 2:3; in the mixed solution B, the volume ratio of the cetyltrimethylammonium bromide aqueous solution to the 2-methylimidazole aqueous solution was 4:3; In the reaction system A, the mass volume ratio of the formate dehydrogenase to all aqueous solutions in the reaction system A is 1-2.5 mg / mL, and the mass ratio of the carbonic anhydrase to formate dehydrogenase is 1:0.5-3.

2. The preparation method according to claim 1, characterized in that In the reaction system A, the mass volume ratio of the formate dehydrogenase to all aqueous solutions in the reaction system A is 2 mg / mL, and the mass ratio of the carbonic anhydrase to formate dehydrogenase is 1:

2.

3. The preparation method according to claim 1, characterized in that In the reaction system A, the concentration of the added cobalt nitrate hexahydrate aqueous solution was 97.5 mmol / L, and the volume ratio of the cobalt nitrate hexahydrate aqueous solution to the 2-methylimidazole aqueous solution was 1:

3.

4. The preparation method according to claim 1, characterized in that In the reaction system B, the concentration of the added zinc nitrate hexahydrate aqueous solution was 97.5 mmol / L, and the volume ratio of the zinc nitrate hexahydrate aqueous solution to the 2-methylimidazole aqueous solution was 1:

3.

5. The use of the hollow ZIF-8 immobilized dual enzyme prepared by the preparation method according to any one of claims 1 to 4, characterized in that: The hollow ZIF-8 immobilized dual enzyme was used for bioelectrocatalytic reduction of CO2 to formic acid, and the concentration of formic acid produced after 180 minutes was 1.11-3.12 mmol / L.

6. The use of the hollow ZIF-8 immobilized dual enzyme according to claim 5, characterized in that: An appropriate amount of deionized water was added to the anode chamber, and a certain amount of hollow ZIF-8 immobilized dual enzyme, 2-hydroxy-1,4-naphthoquinone, and reduced coenzyme I were added to the cathode chamber, wherein the mass volume ratio of the hollow ZIF-8 immobilized dual enzyme was 5-9 mg / mL, the molar concentration of 2-hydroxy-1,4-naphthoquinone was 2.5 mmol / L, the molar concentration of reduced coenzyme I was 3.5 mmol / L, and the molar ratio of 2-hydroxy-1,4-naphthoquinone to reduced coenzyme I was 2.5:3.5; Using 50 mmol / L Tris-HCl buffer with a pH of 6.5 as the reaction solvent, CO2 was bubbled at a flow rate of 100 mL / min, the applied voltage was -0.65 V, and the reference electrode was Ag / AgCl, thereby achieving bioelectrocatalytic CO2 reduction. Water was electrolyzed in the anode chamber to generate protons and electrons. The protons entered the cathode chamber through the proton exchange membrane, and the electrons were captured by 2-hydroxy-1,4-naphthoquinone and NAD + reaction to achieve continuous regeneration of reduced coenzyme I.

7. The use of the hollow ZIF-8 immobilized dual enzyme according to claim 6, characterized in that: After 8 cycles of reaction, the relative activity retention of the hollow ZIF-8 immobilized dual enzyme can reach up to 80.1%.

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