Preparation method of MOF (Metal Organic Framework) immobilized double enzyme and application of MOF immobilized double enzyme in enzyme electro-catalysis CO2 reduction

Abstract

The invention discloses a preparation method of MOF (Metal Organic Framework) immobilized bi-enzyme, which is prepared from an organic ligand and zinc metal salt by a mild water-based method, and FDH (Friction Domain Hypoxia) and CA (Carbonic Acid) are immobilized in an embedding manner to form bi-enzyme cascade, so that the distance between enzymes is shortened, and the diffusion of substances in the reaction process is facilitated. The MOF immobilized bi-enzyme is used for enzyme electrocatalysis CO2 reduction, enzyme electrocatalysis NADH regeneration CO2 reduction is carried out on the MOF immobilized bi-enzyme and a PBS solution containing HNQ and NADH in a three-electrode H-type electrolytic tank composed of a cathode chamber, a proton exchange membrane and an anode chamber, the prepared MOF immobilized bi-enzyme is added into the cathode chamber, water is electrolyzed in the anode chamber to generate protons, the protons enter the cathode chamber through the proton exchange membrane, and the protons enter the anode chamber through the proton exchange membrane. Continuous regeneration of NADH is realized through reaction with NAD < + >, and the rate of cascade reaction is increased by combining substrate enrichment and NADH electrocatalytic regeneration. CO2 is efficiently captured by CA so as to enrich a substrate, and CO2 is efficiently reduced into formate in an enzyme electro-catalysis system in combination with electro-catalysis NADH regeneration.

Description

Technical Field

[0001] The invention belongs to the field of nanomaterials and biotechnology, and specifically relates to a method for preparing a MOF-embedded dual enzyme and an enzyme electrocatalytic CO2 reduction method thereof. Background Art

[0002] As a greenhouse gas, excessive CO2 content in the air is the main cause of global warming. The greenhouse effect leads to frequent extreme weather, causing environmental problems such as land desertification and sea level rise. The large-scale emission of greenhouse gases poses a threat to the Earth's poles and coastal countries. These problems will seriously affect human survival and development. CO2 emission reduction is imminent.

[0003] Enzymatic capture and conversion of CO2 refers to the use of enzyme protein catalysis to catalyze the conversion of CO2 into high value-added products under mild conditions. Enzymatic catalysis of CO2 provides a green and effective alternative for CO2 conversion due to its superior stereospecificity and regiochemical selectivity. Oxidoreductases (such as FDH) can reduce the oxidation state of CO2 and form products such as formate. The conversion of CO2 to formic acid based on FDH (Formate dehydrogenase) has been widely studied because the reaction is simple and the yield is relatively high. The coenzyme NADH-dependent FDH is a monomeric globular protein with one or two identical active sites, which requires NADH or an artificial electron mediator as an electron source for CO2 reduction. NADH regeneration is achieved by electrochemical methods using HNQ (2-hydroxy-1,4-naphthoquinone) as an electron mediator. CA (Carbonic anhydrase) can be used to increase the concentration of CO2 in solution for in vitro conversion through biocatalytic, electrocatalytic, photocatalytic and thermocatalytic processes. CA is also used for enzymatic CO2 reduction with cofactor-dependent FDH and NADH regeneration, improving system performance by increasing CO2 solubility. Currently, CO2 can be used as a carbon source to synthesize high-value-added carbon-containing products and energy compounds such as formic acid, methanol, formaldehyde and methane under different enzyme catalytic conditions, which has become a current research hotspot and achieved efficient utilization of CO2. Bioenzymatic method is a win-win strategy for solving energy and environmental problems.

[0004] MOF (Metal-organic framework) has the characteristics of high specific surface area, high porosity, easy functionalization, and adjustable structure. It is a promising new generation of materials with CO2 adsorption capacity. This MOF is mainly composed of Zn-rich 2+They are made of materials that do not require the aid of catalysts because they have both Lewis acidic and Lewis / Bronsted basic defect sites, which are the main adsorption sites for CO2, while other MOFs only have Lewis acidic defect sites. Enzyme electrocatalytic systems (EES) combine enzyme catalysis and electrochemical technology, promoting the rapid development of bioelectrochemistry. Enzymes convert CO2 into valuable chemicals such as formic acid, methane and methanol in electrocatalytic systems with high activity and selectivity. It is also a win-win strategy for solving energy and environmental problems.

[0005] Encapsulating enzymes in MOF synthesis is a green method for preparing immobilized enzyme catalysts, which provides superior properties, including high enzyme activity retention and adjustable geometric porosity, thereby providing a favorable 3D microenvironment for the enzyme to prevent enzyme denaturation and inactivation. This co-precipitation immobilization method should follow the following principles: first, enzyme denaturation should be minimized during the immobilization process; second, the mass transfer limitation between the enzyme and the substrate in the co-immobilization system should be very low. In addition, for the reduction of CO2, improving the solubility of CO2 in the co-immobilization system is also the key to improving the efficiency of CO2 reduction. Summary of the invention

[0006] In view of the above-mentioned prior art, the present invention provides a method for preparing a MOF-encapsulated dual enzyme and using it for enzyme electrocatalysis of CO2 reduction. FDH and CA dual enzymes are encapsulated in MOF using a mild water-based method, and the MOF-immobilized dual enzyme is placed in the cathode chamber using an enzyme electrocatalysis system to achieve CO2 reduction, so that it is efficiently converted into formate. This method uses MOF materials to encapsulate dual enzymes and is applied to dual enzyme cascade enzyme electrocatalysis to efficiently reduce CO2, shortening the distance between the dual enzymes, and providing a new strategy combining MOF-immobilized dual enzymes and enzyme electrocatalysis.

[0007] In order to solve the above technical problems, the present invention proposes a method for preparing a MOF-immobilized dual enzyme, which mainly uses an organic ligand and a zinc metal salt to prepare a MOF-immobilized dual enzyme by a mild water-based method, wherein the organic ligand is 2,5-dihydroxyterephthalic acid, the zinc metal salt is zinc nitrate, the dual enzyme is FDH and CA, and the MOF-immobilized dual enzyme is recorded as FDH&CA@Zn-MOF-74; the specific process of the above preparation method is as follows;

[0008] The 2,5-dihydroxyterephthalic acid was dissolved in PBS buffer at a mass-to-volume ratio of 5 mg / mL, and then a certain amount of zinc nitrate, FDH and CA were added to obtain a mixture, wherein the molar ratio of 2,5-dihydroxyterephthalic acid to zinc nitrate was 1:3-1:20, the mass concentration of FDH was 0.5-2.5 mg / mL, and the mass concentration of CA was 0.5-2 mg / mL; the mixture was stirred for 30 minutes; the particles were collected by centrifugation, washed with ice water for 30 minutes, and vacuum dried to obtain the MOF-immobilized dual enzyme FDH&CA@Zn-MOF-74.

[0009] Furthermore, the preparation method of the present invention, wherein:

[0010] In the mixture, the molar ratio of the 2,5-dihydroxyterephthalic acid to zinc nitrate is 1:5, the mass concentration of the FDH is 2.0 mg / mL, and the mass concentration of the CA is 1.0 mg / mL.

[0011] The MOF-immobilized dual enzyme prepared by the preparation method of the present invention is used for enzyme electrocatalytic CO2 reduction, mainly by using the MOF-immobilized dual enzyme FDH&CA@Zn-MOF-74 and a PBS solution containing HNQ and NADH to perform enzyme electrocatalytic NADH regeneration and CO2 reduction in an H-type electrolytic cell; the steps are as follows:

[0012] (1) In the cathode chamber, a PBS solution containing HNQ and NADH is first added, and then the MOF-immobilized dual enzyme FDH&CA@Zn-MOF-74 is added, wherein the mass volume ratio of the MOF-immobilized dual enzyme FDH&CA@Zn-MOF-74 to the PBS solution is 2-10 mg / mL, and at the same time, CO2 is bubbled;

[0013] (2) adding deionized water of an equal volume to the PBS solution into the anode chamber; electrolyzing water in the anode chamber to generate protons, which enter the cathode chamber through the proton exchange membrane;

[0014] (3) In the cathode chamber, protons and NAD + The reaction achieves continuous regeneration of NADH and ultimately reduces CO2 to produce formate.

[0015] Furthermore, the pH of the PBS solution is 5.5-7.5, preferably, the pH of the PBS solution is 6; the concentration of the HNQ is 1-5 mmol / L, preferably, the concentration of the HNQ is 3 mmol / L; the concentration of the NADH is 1-5 mmol / L, preferably, the concentration of the NADH is 4 mmol / L; and the ventilation rate of CO2 is 100 mL / min.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The MOF-immobilized dual enzyme prepared by the present invention, wherein the two enzymes are immobilized by co-encapsulation, shortens the distance between the enzymes, facilitates the diffusion of substances during the reaction, and combines substrate enrichment and NADH electrocatalytic regeneration to increase the rate of the cascade reaction.

[0018] (2) The addition of CA greatly promotes the capture of CO2, thereby accelerating its further conversion into formate under the catalysis of FDH.

[0019] (3) Since MOF has high enzyme activity retention rate and adjustable geometric porosity, MOF is used to encapsulate enzymes to provide a favorable 3D microenvironment for enzymes to prevent enzyme denaturation and inactivation

[0020] (4) The prepared FDH&CA@Zn-MOF-74 has a maximum formate yield of 3.01 mmol / L in the enzyme electrocatalytic CO2 reduction system and can also be reused.

[0021] In summary, the MOF-immobilized dual enzyme prepared by the present invention can effectively solve the problems of easy inactivation, low activity, and poor stability of biological enzymes, and can be used for efficient enzyme electrocatalytic CO2 reduction. Compared with free enzymes, the MOF-immobilized dual enzyme prepared by encapsulating FDH and CA in MOF solves the problems of poor stability and non-reusability of the free enzyme FDH. CA is used to efficiently capture CO2 to enrich the substrate, and then combined with electrocatalytic NADH regeneration, CO2 is efficiently reduced to formate in the enzyme electrocatalytic system. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1-1 It is a roadmap for the preparation of FDH&CA@Zn-MOF-74 using a mild water-based method;

[0023] Figure 1-2 yes Figure 1-1 Schematic diagram of the prepared FDH&CA@Zn-MOF-74 for enzymatic electrocatalytic CO2 reduction;

[0024] Figure 2-1 is a scanning electron microscope image of Zn-MOF-74 during the preparation process of Example 1;

[0025] Figure 2-2 is a scanning electron microscope image of FDH&CA@Zn-MOF-74 prepared in Example 1;

[0026] Figure 3 is the XRD pattern of Zn-MOF-74 and the prepared FDH&CA@Zn-MOF-74 in the preparation process of Example 1;

[0027] Figure 4 The SDS-PAGE results of the relevant materials (FDH, CA, FDH&CA on Zn-MOF-74) and the prepared FDH&CA@Zn-MOF-74 in the preparation process of Example 1;

[0028] Figure 5 The FDH&CA@Zn-MOF-74 prepared in Example 1 was used for enzyme electrocatalytic CO2 reduction, and the amperometric curves under different HNQ concentrations were obtained;

[0029] Figure 6-1 The FDH&CA@Zn-MOF-74 prepared in Example 1 was used for enzyme electrocatalytic CO2 reduction, and the amperometric curves under different NADH concentrations were obtained;

[0030] Figure 6-2 The FDH&CA@Zn-MOF-74 prepared in Example 1 was used for enzyme electrocatalytic CO2 reduction, and the formate yields under different NADH concentrations;

[0031] Figure 7 Example 5 is the yield of formate under different ratios of linker (H4DOBDC) and metal ion (zinc nitrate);

[0032] Figure 8-1 The effect of different CA concentrations on formate formation in Example 6;

[0033] Figure 8-2 The effect of different CA concentrations on current density in Example 6;

[0034] Figure 8-3 The effect of different FDH concentrations on formate formation in Example 6;

[0035] Figure 8-4 The effect of different FDH concentrations on current density in Example 6;

[0036] Fig. 9 The effect of different amounts of FDH&CA@Zn-MOF-74 added to the cathode chamber in Example 7 on the amount of formate produced;

[0037] Fig.10 : is the formate yield in different enzymatic reaction systems of Example 8, wherein the solid symbols indicate NADH regeneration, and the hollow symbols indicate no NADH regeneration. DETAILED DESCRIPTION

[0038] The design idea of ​​the preparation method of a MOF-immobilized dual enzyme proposed in the present invention is: at room temperature, a mild water-based method is used to encapsulate FDH and CA into Zn-MOF-74 during the formation of MOF, so as to protect the spatial structure of the enzyme molecules from being destroyed. The above-mentioned MOF-immobilized dual enzyme is used for enzyme electrocatalytic CO2 reduction using an enzyme electrocatalytic system, so that it can be efficiently converted into formate. This method uses MOF materials to embed dual enzymes and is applied to dual-enzyme cascade enzyme electrocatalysis to efficiently reduce CO2. Enzyme electrocatalysis is carried out in a three-electrode H-type electrolytic cell consisting of a cathode chamber, a proton exchange membrane, and an anode chamber to regenerate NADH and reduce CO2. The prepared MOF-immobilized dual enzyme is added to the cathode chamber, and water is electrolyzed in the anode chamber to generate protons. The protons enter the cathode chamber through the proton exchange membrane and react with NAD + The reaction achieves continuous regeneration of NADH. Ultimately, CO2 can be reduced to form formate. The present invention synthesizes Zn-MOF-74 in a mild aqueous phase at room temperature, and immobilizes FDH and CA in an embedding manner to form a dual enzyme cascade, shortening the distance between the dual enzymes, which is beneficial to the diffusion of substances during the reaction, and combining substrate enrichment and NADH electrocatalytic regeneration to increase the rate of the cascade reaction. CA is used to efficiently capture CO2 to enrich the substrate, and then combined with electrocatalytic NADH regeneration to enable CO2 to be efficiently reduced to formate in the enzyme electrocatalytic system. The present invention provides a new strategy combining MOF immobilized dual enzymes and enzyme electrocatalysis.

[0039] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the following embodiments are by no means limiting the present invention in any way.

[0040] Example 1

[0041] Preparation of a MOF-immobilized dual enzyme and its application in enzymatic electrocatalytic CO2 reduction.

[0042] like Figure 1-1 As shown, an organic ligand and a zinc metal salt are used, the organic ligand is 2,5-dihydroxyterephthalic acid (H4DOBDC), the zinc metal salt is zinc nitrate, and a mild water-based method is used to prepare the MOF-immobilized dual enzyme, and the dual enzyme (FDH and CA) is embedded in the MOF preparation process. The specific process is:

[0043] 40.0 mg (0.202 mmol) of yellow solid H4DOBDC was dissolved in 8.0 mL of PBS buffer (50 mM, pH = 6). The solution was then mixed with 8.0 mL of PBS buffer containing zinc nitrate (0.126 M) and 2.0 mg / mL of FDH and 1.0 mg / mL of CA. The resulting mixture was stirred for 30 min. Finally, the synthesized particles were collected by centrifugation (12 000 r / min), washed, stirred and washed with ice water for 30 min, and vacuum dried to obtain the MOF-immobilized dual enzyme, recorded as FDH&CA@Zn-MOF-74.

[0044] like Figure 2-2 As shown, the FDH&CA@Zn-MOF-74 prepared in Example 1 was subjected to scanning electron microscopy. Figure 2-1 It shows that Zn-MOF-74 presents a complete rod-like structure with a size of 8×2×1μm, and the morphology of FDH&CA@Zn-MOF-74 is similar to that of the original sample without enzyme.

[0045] like Figure 3 As shown, the FDH&CA@Zn-MOF-74 prepared in Example 1 was characterized by XRD ray diffraction. Zn-MOF-74 and FDH&CA@Zn-MOF-74 biocomposites showed obvious diffraction peaks at 2θ=6.8° and 11.8°, corresponding to the (110) and (300) crystal planes, respectively, proving the successful synthesis of the carrier. In addition, there is no significant difference between the crystal structures of the two, indicating that the carrier still maintains a good crystal structure after the enzyme is immobilized.

[0046] The FDH&CA@Zn-MOF-74 prepared above was used for enzyme electrocatalytic CO2 reduction, such as Figure 1-2 As shown,

[0047] 80 mg of FDH&CA@Zn-MOF-74 was added to the cathode chamber of a PBS solution containing 3 mmol / L HNQ and 4 mmol / L NADH, and CO2 was bubbled at the same time. The CO2 aeration rate was 100 mL / min, and deionized water of the same volume as the PBS solution was added to the anode chamber. In the cathode chamber, CA can accelerate the CO2 hydration rate, and FDH uses NADH as a reducing agent to reduce CO2 to formic acid, and HCO 3- It can also be used as a substrate for the production of formic acid. This reaction is usually carried out in a buffer solution, so the product can be HCOOH / HCOO - The anode chamber electrolyzes water to produce protons, which enter the cathode chamber through the proton exchange membrane and react with NAD +The reaction achieves continuous regeneration of NADH. High performance liquid chromatography is subsequently used to detect the formation of formate. The solubility of CO2 in water is increased by adsorbing CO2 with CA, and the electrocatalytic NADH regeneration is enhanced by using electron mediators, effectively reducing CO2 to formate in the enzyme electrocatalytic system. High performance liquid chromatography is subsequently used to detect the formation of formate.

[0048] Example 2

[0049] Protein electrophoresis characterization of FDH&CA@Zn-MOF-74 prepared in the present invention.

[0050] The comparative sample FDH&CA on Zn-MOF-74 was prepared by physically mixing the Zn-MOF-74 carrier with the FDH and CA solutions and shaking for 1 min to obtain FDH&CA on Zn-MOF-74.

[0051] The comparative sample FDH&CA on Zn-MOF-74 and the FDH&CA@Zn-MOF-74 sample prepared in Example 1 were washed with deionized water several times, centrifuged, and then loaded onto SDS-PAGE gel for qualitative protein analysis. Figure 4 As shown, free CA (lane 1) and FDH (lane 2) showed obvious bands at 29 kDa and 45 kDa, the test sample FDH&CA@Zn-MOF-74 (lane 3) showed corresponding bands at 29 kDa and 45 kDa, while the FDH&CA on Zn-MOF-74 sample (lane 4) had no bands. This experiment clearly showed that FDH and CA were indeed encapsulated inside Zn-MOF-74, and the encapsulated enzyme molecules were not easily removed from the carrier. On the contrary, the enzyme molecules were simply removed from the surface of FDH&CA on Zn-MOF-74 because they were only physically adsorbed on the MOF surface.

[0052] Example 3

[0053] Optimization of HNQ concentration during enzymatic electrocatalytic CO2 reduction using FDH&CA@Zn-MOF-74.

[0054] The FDH&CA@Zn-MOF-74 prepared in Example 1 was used for enzymatic electrocatalytic CO2 reduction. The reduction process was basically the same as that in Example 1, except that the reaction was performed six times according to the amount of HNQ added. The amount of HNQ added each time was 0 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L and 5 mmol / L, respectively, and 6 amperometric curves were finally obtained. Figure 5The ampere curves under different concentrations of HNQ are shown in Figure 2. When no HNQ is added, the current is very small and almost zero, and there is almost no electron transfer in the reaction system. However, in the HNQ system with different concentrations, the current always exists. This shows that HNQ can act as an electron medium to transfer electrons from the cathode to the reaction system. It can be seen that when the addition amount of HNQ is 3mmol / L, the electron transfer amount is the largest.

[0055] Example 4

[0056] FDH&CA@Zn-MOF-74 was used to optimize the NADH concentration during enzymatic electrocatalytic CO2 reduction.

[0057] The FDH&CA@Zn-MOF-74 prepared in Example 1 was used for enzymatic electrocatalytic CO2 reduction. The reduction process was basically the same as that in Example 1, except that the reaction was performed six times according to the amount of NADH added. The amount of NADH added each time was 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L and 5 mmol / L, respectively. Finally, six amperometric curves and formate yields were obtained. Figure 6-1 and Figure 6-2 The amperometric curves at different NADH concentrations and formate concentrations are shown respectively. Figure 6-1 It shows that the current and electron transfer amount increase with the increase of NADH concentration. This may be because with the increase of NADH concentration, the rate of enzyme-catalyzed CO2 reduction reaction is accelerated, the NADH consumption increases, and NAD + The increase in production causes HNQ to transfer to NAD + More electrons are transferred to regenerate NADH. When the NADH concentration is 5mmol / L, the current is very close to 4mmol / L. This is because the amount of immobilized enzyme added is fixed and the reaction rate reaches saturation. Figure 6-2 It shows that the formate production increases with the increase of NADH concentration. When the added NADH is 5mmol / L, the generated formate no longer increases and the reaction reaches saturation, indicating that the addition of 4mmol / L NADH is sufficient. It can be seen that when the NADH concentration is 4mmol / L, the formate reaches saturation, and the maximum formate concentration is 1.166mmol / L.

[0058] Example 5

[0059] Optimization of the ratio of organic ligand H4DOBDC and zinc nitrate in the preparation of FDH&CA@Zn-MOF-74.

[0060] The preparation method is basically the same as that of Example 1, except that when preparing FDH&CA@Zn-MOF-74, five samples were prepared by changing the ratio of H4DOBDC to zinc nitrate, and the ratio of H4DOBDC to zinc nitrate in each sample was 1:3, 1:5, 1:10, 1:15, and 1:20, respectively. The five FDH&CA@Zn-MOF-74 samples prepared above were used for enzyme electrocatalytic CO2 reduction. Figure 7 The yield of formate from the samples prepared with different ratios of H4DOBDC and zinc nitrate for enzymatic electrocatalytic CO2 reduction is shown. Figure 7 It shows that the activity of the enzyme increases first and then decreases. When the ratio of the linker H4DOBDC to the zinc nitrate metal ion is 1:5, the formate production is the highest, so the enzyme activity is the highest. It can be seen that when the ratio of the linker to the metal ion is 1:5, the formate production reaches the highest, and the maximum formate is 1.181mmol / L.

[0061] Example 6

[0062] In the preparation of FDH&CA@Zn-MOF-74, the concentrations of FDH and CA were optimized.

[0063] The preparation method is basically the same as that in Example 1, except that, when preparing FDH&CA@Zn-MOF-74, the concentration of FDH is set to 0.5 mg / mL, and five samples are made according to the amount of CA added. The amount of CA added to each sample is 0 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL and 2 mg / ml respectively; the concentration of CA is set to 1 mg / mL, and five samples are made according to the amount of FDH added. The amount of FDH added to each sample is 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL and 2.5 mg / ml respectively. Figure 8-1 and Figure 8-2 are respectively the formate concentration and current density at different CA addition ratios. In this embodiment, Figure 8-1 It can be seen that when the CA concentration increases to 1.5 mg / mL, the reaction rate decreases and the current density also decreases ( Figure 8-2 ), this is because when the concentration of CA is low, the hydration reaction of CO2 is slower than the formic acid synthesis reaction catalyzed by FDH, and is the rate-limiting step of the coupling reaction. When the concentration of CA is increased, the hydration of CO2 is accelerated, but due to the limited concentration of FDH, the enzyme-catalyzed formic acid synthesis becomes the rate-limiting step. Figure 8-3 and Figure 8-4They are formate concentration and current density at different FDH addition ratios. It can be seen that increasing the concentration of FDH added during immobilization can effectively increase the overall rate of the coupling reaction. This is because FDH can catalyze the reduction of CO2 to formic acid. Properly increasing the catalyst concentration can accelerate the CO2 reduction rate. When the FDH concentration increases from 2 mg / mL to 2.5 mg / mL, the amount of formic acid generated decreases, and the current density also decreases ( Figure 8-4 ). This may be because the two enzymes compete for the limited space in the carrier at higher concentrations. It can be seen that when the FDH concentration is 2 mg / mL and the CA concentration is 1 mg / mL, the formate production reaches the highest, and the maximum formate is 2.25 mmol / L.

[0064] Example 7

[0065] The amount of immobilized dual enzymes added was optimized in the process of enzymatic electrocatalytic CO2 reduction using FDH&CA@Zn-MOF-74.

[0066] The FDH&CA@Zn-MOF-74 prepared in Example 1 was used for enzyme electrocatalytic CO2 reduction. The reduction process was basically the same as that in Example 1, except that the amount of FDH&CA@Zn-MOF-74 added was different, and it was done five times. The amount of FDH&CA@Zn-MOF-74 added each time was: 20 mg, 40 mg, 60 mg, 80 mg and 100 mg. The volume of the mixed solution in the cathode chamber was 10 mL. Fig. 9 is the process curve of formate production with different addition amounts of immobilized dual enzymes. Fig. 9 As shown in the figure, with the increase of the amount of immobilized enzyme, the catalyst in the system increases, and thus the production of formate gradually increases. When the amount of immobilized enzyme is 80 mg, the formate production tends to the maximum value, and the reaction reaches saturation. Therefore, 80 mg is selected as the optimal addition amount of immobilized enzyme in the reaction system. It can be seen that when the amount of immobilized enzyme added is 80 mg, the formate production reaches saturation, and the maximum formate is 3.01 mmol / L.

[0067] Example 8

[0068] Comparison of different enzymatic CO2 reduction systems.

[0069] Six different enzymatic reaction systems were studied for the electrochemical regeneration of NADH and the yield of CO2 reduction to formate by MOF-immobilized dual enzymes. Fig.10 In the figure, solid symbols indicate that NADH is regenerated; hollow symbols indicate that NADH is not regenerated. Fig.10As shown in the figure, the free enzyme generates a maximum of 0.503 mmol / L formate within 3 hours. Encapsulating FDH in Zn-MOF-74 alone and adding NADH regeneration can increase the amount of formate generated. The FDH@Zn-MOF-74 coupled NADH regeneration system generates 1.45 mmol / L formate within 3 hours. This is because the enrichment of CO2 by Zn-MOF-74 accelerates the reduction reaction of CO2, and the addition of NADH regeneration can provide a continuous source of reducing equivalents for CO2 reduction. Further, the dual enzyme FDH and CA are co-encapsulated in Zn-MOF-74, and the resulting MOF-immobilized dual enzyme FDH&CA@Zn-MOF-74 system is combined with electrochemical NADH regeneration, generating a maximum of 3.01 mmol / L formate in 3 hours. This is because CA promotes the capture of CO2 and accelerates the hydration of CO2, thereby enriching more substrates in the reaction system and accelerating the forward reaction catalyzed by FDH. In addition, the co-encapsulation of two enzymes can shorten the spatial distance between the two enzymes, thereby achieving an efficient enzyme cascade reaction. In addition, the electrochemical regeneration of NADH can maintain the concentration of NADH and relieve the + The inhibition of NADH is beneficial to the formation of formate. The immobilized enzyme alone or the electrocatalytic NADH regeneration system has a significant improvement in formate production compared to the pure free enzyme system. In summary, the formate production of the MOF-immobilized dual enzyme FDH&CA@Zn-MOF-74 in the enzyme electrocatalytic CO2 reduction system is 6 times that of the free enzyme system.

[0070] 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, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can make many modifications without departing from the purpose of the present invention, all of which are within the protection of the present invention.

Claims

1. A method for preparing a MOF-immobilized dual enzyme, characterized in that: The MOF-immobilized dual enzyme was prepared by a mild water-based method using an organic ligand and a zinc metal salt. The organic ligand was 2,5-dihydroxyterephthalic acid, the zinc metal salt was zinc nitrate, the dual enzyme was FDH and CA, and the MOF-immobilized dual enzyme was recorded as FDH&CA@Zn-MOF-74.

2. The preparation method according to claim 1, characterized in that: The specific process is: dissolving the 2,5-dihydroxyterephthalic acid in PBS buffer at a mass-to-volume ratio of 5 mg / mL, and then adding a certain amount of zinc nitrate, FDH and CA to obtain a mixture, wherein the molar ratio of 2,5-dihydroxyterephthalic acid to zinc nitrate is 1:3-1:20, the mass concentration of FDH is 0.5-2.5 mg / mL, and the mass concentration of CA is 0.5-2.0 mg / mL; stirring the above mixture for 30 minutes; collecting the particles by centrifugation, stirring and washing with ice water for 30 minutes, and vacuum drying to obtain the obtained product, which is MOF-immobilized dual enzyme FDH&CA@Zn-MOF-74.

3. The preparation method according to claim 2, characterized in that: In the mixture, the molar ratio of the 2,5-dihydroxyterephthalic acid to zinc nitrate is 1:5, the mass concentration of the FDH is 2.0 mg / mL, and the mass concentration of the CA is 1.0 mg / mL.

4. The use of the MOF-immobilized dual enzyme prepared by the preparation method according to any one of claims 1 to 3, characterized in that: The MOF-immobilized dual enzymes were used for enzymatic electrocatalytic CO2 reduction.

5. The use of MOF-immobilized dual enzyme according to claim 4, characterized in that: The MOF-immobilized dual enzyme FDH&CA@Zn-MOF-74 and a PBS solution containing HNQ and NADH are used to perform enzyme electrocatalysis to regenerate and reduce CO2 in an H-type electrolytic cell; the steps are as follows: In the cathode chamber, a PBS solution containing HNQ and NADH is first added, and then the MOF-immobilized dual enzyme FDH&CA@Zn-MOF-74 is added, wherein the mass volume ratio of the MOF-immobilized dual enzyme FDH&CA@Zn-MOF-74 to the PBS solution is 2-10 mg / mL, and CO2 is bubbled at the same time; In the anode chamber, deionized water of the same volume as the PBS solution is added; the anode chamber electrolyzes water to generate protons, and the protons enter the cathode chamber through the proton exchange membrane; In the cathode chamber, protons react with NAD + The reaction achieves continuous regeneration of NADH and ultimately reduces CO2 to produce formate.

6. The use of MOF-immobilized dual enzyme according to claim 5, characterized in that: The pH of the PBS solution is 5.5-7.5, the concentration of the HNQ is 1-5 mmol / L, the concentration of the NADH is 1-5 mmol / L; the ventilation rate of CO2 is 100 mL / min.

7. The use of MOF-immobilized dual enzyme according to claim 5, characterized in that: The pH of the PBS solution is 6, the concentration of HNQ is 3 mmol / L, the concentration of NADH is 4 mmol / L; and the ventilation rate of CO2 is 100 mL / min.

Images