Preparation method and application of rhodium-based electron medium and enzyme co-loaded COF photocatalyst

Abstract

The invention provides a preparation method of a rhodium-based electron medium and enzyme co-loaded COF photocatalyst, and provides a two-in-one synthesis method, by means of the method, the rhodium-based electron medium is successfully modified on a COF frame, efficient transfer of photo-induced electrons is achieved, the regeneration rate of NADH is increased, meanwhile, FDH enzyme is co-loaded, the FDH enzyme is embedded into the COF frame, and the catalytic activity of the COF frame is improved. The stability and reusability of enzyme reaction in the system are effectively protected, efficient utilization of NADH is achieved, the performance of reducing carbon dioxide through the photo-enzyme cascade reaction is remarkably enhanced, and the excellent photo-enzyme cascade performance is shown. When the loading and adding mass ratio of the rhodium-based electron medium is 0.6, the yield of formic acid per unit enzyme obtained by the loading enzyme reaction is 9 times of the yield of formic acid per unit enzyme reaction of the free enzyme reaction, which indicates that the material disclosed by the invention has higher photocatalytic ability and enzyme-retaining activity.

Description

Technical Field

[0001] The invention belongs to the field of photocatalysis, and specifically relates to a method for preparing a photocatalyst loaded with rhodium COF (covalent organic framework) and an application of realizing NADH coenzyme regeneration and cascading with a FDH (formate dehydrogenase) catalytic hydrogenation system for reducing carbon dioxide to formate. Background Art

[0002] Over the past 100 years, the world's energy demand has increased year by year, and about 80% of energy consumption is based on fossil fuels, including coal, oil and natural gas. By the middle of the 21st century, global energy consumption is expected to increase by more than 50%. Since fossil fuels are limited and regional on a global scale, it is a huge challenge to ensure that this demand can be met in the face of other potential problems such as energy supply. Due to the use of fossil fuels, large amounts of emissions such as CO2, SO2 and oxide particles are the main causes of global warming and environmental pollution. Therefore, it is urgent to develop a new energy transformation to reduce global CO2 emissions. Biocarbon fixation is considered to be one of the most economical, green and reliable ways to fix CO2. It has the advantages of being recyclable and having few side effects, and has attracted widespread attention from scientists. As a natural biocatalyst, formate dehydrogenase (FDH) can convert CO2 into liquid chemical formic acid, one of the main raw materials for organic chemical industry, under the catalysis of reduced cofactor nicotinamide adenine dinucleotide (NADH). Solar energy is a clean, pollution-free, sustainable green energy. Under the action of photosensitizers, it can regenerate NADH to drive enzymes to catalyze CO2 reduction. It has attracted widespread attention for its ability to achieve green synthesis of high value-added chemicals.

[0003] In addition, CO2 conversion with FDH often requires the provision of a stoichiometric amount of nicotinamide cofactor (NADH) to achieve catalytic turnover, which brings operational complexity, high cost, and application limitations. Therefore, it is of great significance to develop efficient NADH regeneration strategies, among which the combination of rhodium-based electron mediators Cp*Rh(bpy)H2O (Cp*=pentamethylcyclodiene; bpy=2,2′-bipyridine) is an effective way to achieve NADH regeneration. Therefore, the cascade reaction of enzyme-catalyzed reactions and artificial NADH regeneration provide a feasible route for high-performance photocatalytic CO2 reduction. At present, the cost of large-scale production of FDH by microbial expression is still high, and its practical application often suffers from high cost, non-reusability, and poor stability under harsh conditions. The above problems can be solved by integrating enzymes and NADH regeneration photocatalysts in porous solid supports to produce heterogeneous cascade biocatalysts.

[0004] In recent years, covalent organic frameworks (COFs) with long-term water stability and metal-free skeletons have emerged as a new generation of porous solid supports and have been developed as environmentally friendly platforms with high loading and operational stability, which can protect biocatalysts from deactivation, facilitate product purification, and significantly enhance stability and reusability. Summary of the invention

[0005] Based on the above research background, we created a "two-in-one" strategy: the electron mediator and FDH are co-anchored in the pores of imine-linked COF to construct an integrated platform for heterogeneous CO2 photoconversion to formate. The rhodium-based electron mediator Cp*Rh(bpy)H2O can be modified on the COF backbone via coordination bonds for NADH photoregeneration and FDH is embedded in the COF channel to form an integrated photocatalyst-enzyme coupling system. This step-by-step assembled photoenzyme system exhibits excellent operational stability and recyclability, exceeding the standards of state-of-the-art photobiocoupled artificial photosynthesis systems. Using this approach, we achieved highly selective photocatalytic conversion of CO2 to formate.

[0006] This method not only successfully modified the rhodium-based electron mediator on the COF framework, realized the efficient transfer of photogenerated electrons, and increased the regeneration rate of NADH, but also co-loaded the FDH enzyme and embedded the FDH enzyme into the COF framework, effectively protecting the stability and reusability of the enzyme reaction in the system, achieving efficient utilization of NADH, and significantly enhancing the performance of the photoenzyme cascade reaction to reduce carbon dioxide, showing excellent photoenzyme cascade performance. The specific scheme is as follows: Step 1) preparing a photocatalyst, denoted as COF, with 1,2,3-trialdehyde phloroglucinol as the donor and electron-deficient 2,2'-bipyridine-6,6'-diamine as the acceptor at a molar ratio of 1:2 between the donor and the acceptor; Step 2) mixing the COF obtained in step 1) with a methanol solution containing a certain amount of rhodium-based electron mediator [Rh(Cp*)C12]2, wherein the amount of [Rh(Cp*)C12]2 in the methanol solution is determined according to the mass ratio of the Rh content therein to the COF; stirring the mixed solution for 24 hours to modify the rhodium electron mediator into the COF, and the resulting product is a rhodium-based electron mediator COF photocatalyst, which is recorded as photocatalyst COF-Rh; Step 3) The COF-Rh prepared in step 2) and 5 mg of FDH were dissolved in 2 mL of phosphate buffer solution (PBS), reacted for 3 h, centrifuged and washed, and freeze-dried to obtain a COF framework material loaded with both electron mediators and enzymes, recorded as photocatalyst COF-Rh@FDH.

[0007] Further, it is characterized in that in the step 1), 0.0504 g of 1,2,3-trialdehyde phloroglucinol and 0.0671 g of 2,2'-bipyridine-6,6'-diamine are mixed in a molar ratio of 1:2 to obtain a mixture, and the mixture is added to 15 mL of solution A, wherein solution A is obtained by mixing o-dichlorobenzene and N,N-dimethylacetamide in a volume ratio of 1:3. After ultrasonic mixing for 10 minutes, 5 mL of acetic acid solution (6M) is added and ultrasonicated for 5 minutes. Then, the freezing-vacuuming-thawing process is repeated three times, and the reaction is carried out at 120°C for three days. After filtering and separation, the precipitate is washed with tetrahydrofuran, ultrapure water and methanol in turn, and dried in a vacuum oven at 60°C, and the resulting product is the COF.

[0008] Furthermore, it is characterized in that in the step 2), the COF prepared in step 1) and the methanol solution containing [Rh(Cp*)C12]2 are added to the reactor at a mass ratio of 0.4, 0.6, 0.8, and 1.0, and the reaction is carried out at a speed of 240 for 24 hours. After the reaction is completed, the solid is filtered and separated, and the solid is washed with ultrapure water and methanol in turn. After drying, the obtained product is the photocatalyst COF-Rh.

[0009] Furthermore, it is characterized in that in the step 3), the COF-Rh and FDH enzymes prepared in step 2) are added to a PBS buffer solution according to a mass of 0.5, and reacted at room temperature for 3 hours. After the reaction, the product is centrifuged, washed three times with a PBS solution, and then placed in a freeze dryer, freeze-dried for 24 hours, and placed in a freezing layer for standby use, which is called COF-Rh@FDH.

[0010] Furthermore, the COF-Rh@FDH prepared by the present invention adopts a "two-in-one" strategy: the electron medium and FDH are co-anchored in the pores of the imine-linked COF to construct an integrated platform for heterogeneous CO2 photoconversion into formic acid. + , triethanolamine (TEOA) and PBS were added to the reaction system at a mass ratio of 2:1.45:20 and a volume ratio of 20:1. The mixed solution of the above system was reacted for 30 minutes under dark conditions to achieve sufficient mixing and adsorption. Then it was irradiated with a xenon lamp, a 420nm filter was used, and samples were taken at intervals. After filtering with a 0.22um filter head, HPLC was used to detect the formic acid product. The final calculated yield was 30mmol g -1 .

[0011] The present invention has the following benefits: The present invention successfully synthesized a COF containing a bipyridine structure, providing a prerequisite for the subsequent loading modification of a rhodium-based electronic medium.

[0012] The present invention adopts a "two-in-one" approach to load the FDH enzyme while loading a rhodium-based electron medium. The loading modification of the rhodium-based electron medium enhances the photocurrent of the COF, realizes the rapid separation and application of photogenerated electrons, and improves the photocatalytic performance of the material.

[0013] The photocatalytic material synthesized by the present invention has a greatly improved NADH reduction effect after modifying the electron medium, and at the same time, the FDH enzyme utilization rate in the product is high, indicating that the 1,4-NADH product accounts for a high proportion and effectively reduces NAD + .

[0014] The COF-Rh@FDH prepared by the present invention realizes the photoenzyme cascade reaction by loading the enzyme. Not only the NADH obtained by the photoreaction is fully utilized, but also the substrate is efficiently reduced to obtain formic acid with significant yield. It also provides ideas and application cases for other enzyme loadings with negative charges. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the SEM image of the photocatalyst COF-Rh obtained in Example 1; Figure 2 This is the SEM image of the photocatalyst COF-Rh-FDH obtained in Example 2; Figure 3 The XRD patterns of the two photocatalysts COF and COF-Rh obtained in Comparative Example 1 and Example 1 at 0-80°; Figure 4 It is a comparison chart of the reduction rates of the two photocatalysts COF obtained in Comparative Example 1 and Example 1 and COF-Rh with different rhodium-based electron medium loadings on the photocatalytic regeneration of NADH; Figure 5 Fourier infrared peak images of the two photocatalysts COF and COF-Rh obtained in Comparative Example 1 and Example 1; Figure 6 This is a comparison chart of the formic acid content produced per unit enzyme reduction of COF-Rh@FDH and COF-Rh plus free enzyme obtained in Example 2 and Implementation 1; Figure 7 This is a comparison diagram of the impedance of the two photocatalysts, COF and COF-Rh, obtained in Comparative Example 1 and Example 1; Figure 8 The photocurrent response spectra of the two photocatalysts COF and COF-Rh obtained in Comparative Example 1 and Example 1; DETAILED DESCRIPTION

[0016] The present invention will be described in more detail below through specific embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0017] Embodiment 1: COF material preparation: 0.0504 g of 1,2,3-trialdehyde phloroglucinol and 0.0671 g of 2,2'-bipyridine-6,6'-diamine were mixed in a molar ratio of 1:2 to obtain a mixture, and the mixture was added to 15 mL of solution A, which was obtained by mixing o-dichlorobenzene and N,N-dimethylacetamide in a volume ratio of 1:3. After ultrasonic mixing for 10 minutes, 5 mL of acetic acid solution (6M) was added and ultrasonicated for 5 minutes. Then, the freezing-vacuuming-thawing process was repeated three times, and the reaction was carried out at 120°C for three days. After filtering and separation, the precipitate was washed with tetrahydrofuran, ultrapure water and methanol in turn, and dried in a vacuum oven at 60°C, and the obtained product was the COF.

[0018] COF-Rh material preparation: [Rh(Cp*)C12]2 and COF were weighed and mixed according to the mass ratio of 0.4, 0.6, 0.8 and 1.0, added into 2 mL of methanol solution, and reacted at a speed of 240 for 24 hours. After the reaction was completed, the solid was filtered and separated. The solid was washed with ultrapure water and methanol in turn, and after drying, the obtained product was the photocatalyst COF-Rh.

[0019] Embodiment 2: COF-Rh@FDH material preparation: The FDH enzyme and the COF-Rh prepared in Example 1 were added to a PBS buffer solution according to a mass of 0.5, and reacted at room temperature for 3 hours. The product after the reaction was centrifuged, washed three times with a PBS solution, and then placed in a freeze dryer, freeze-dried for 24 hours, and placed in a freezing layer for standby use to prepare COF-Rh@FDH.

[0020] Comparative Example 1: 0.0504 g of 1,2,3-trialdehyde phloroglucinol and 0.0671 g of 2,2'-bipyridine-6,6'-diamine were mixed in a molar ratio of 1:2 to obtain a mixture, and the mixture was added to 15 mL of solution A, which was obtained by mixing o-dichlorobenzene and N,N-dimethylacetamide in a volume ratio of 1:3. After ultrasonic mixing for 10 minutes, 5 mL of acetic acid solution (6M) was added and ultrasonicated for 5 minutes. Then, the freezing-vacuuming-thawing process was repeated three times, and the reaction was carried out at 120°C for three days. After filtering and separation, the precipitate was washed with tetrahydrofuran, ultrapure water and methanol in turn, and dried in a vacuum oven at 60°C, and the obtained product was the COF.

[0021] Tests and Results The activity investigation method of photocatalytic regeneration of NADH provided by the present invention is as follows: Take 2mg composite photocatalyst, 20mg βNAD+, measure 20mL 100mM phosphate buffer (pH=6.8), 1mL triethanolamine (TEOA), choose whether to add 28uL electron mediator M solution according to experimental conditions, and add the mixed solution to the quartz photocatalytic tube. Stir the reaction system magnetically for 30min in the dark, and take a sample as the initial concentration of NADH regeneration. Subsequently, turn on a 300W xenon lamp equipped with a 420nm filter to simulate visible light for photocatalytic NADH regeneration reaction. Take 200uL of samples in a centrifuge tube at regular intervals, add 800uL of deionized water for dilution and centrifuge, take the supernatant and remove the suspended catalyst with a filter head. The resulting clear night is measured by a UV-visible spectrophotometer for absorbance at 340nm, and the reproduction rate of NADH is calculated; The formic acid production investigation method provided by the present invention is as follows: Take 2mg composite photocatalyst, 20mg βNAD+, measure 20mL 100mM phosphate buffer (pH=6.8), 1mL triethanolamine (TEOA), 1.45g NaHCO3, choose whether to add 28uL electron mediator M solution according to experimental conditions, and add the mixed solution to the quartz photocatalytic tube. Stir the reaction system magnetically for 30min under dark conditions, and take a sample as the initial concentration of NADH regeneration. Then, turn on a 300W xenon lamp equipped with a 420nm filter to simulate visible light for photocatalytic NADH regeneration reaction, take 200uL samples at regular intervals, filter them, add them to a liquid phase vial, and then put them into HPLC to analyze the peaks. Then calculate the final yield by comparing with the standard curve. Calculate the amount of formic acid reduction corresponding to the unit enzyme according to the amount of enzyme added to the reaction.

[0022] Figure 1 This is a SEM image of the photocatalyst COF-Rh obtained in Example 1; from the image, it can be seen that the synthesized material has an obvious layered structure, and the entire material structure is compact and regular; Figure 2 This is a SEM image of the photocatalyst COF-Rh-FDH obtained in Example 2; the porous structure of the material can be clearly seen from the image, and the layered structure of the material is still clearly visible after loading the enzyme, indicating that the material structure is highly stable; Figure 3 The XRD diagrams of the two photocatalysts COF and COF-Rh obtained in Comparative Example 1 and Example 1 at 0-80°; from the figure, clear COF characteristic peaks, 100 crystal planes and 210 crystal planes can be seen at 0-5°, and from the comparison diagram of COF and COF-Rh, it can be seen that the crystal form of COF is not changed after loading the rhodium-based electron medium; Figure 4This is a comparison chart of the reduction rate of photocatalytically regenerated NADH by COF obtained in Comparative Example 1 and Example 1 and COF-Rh with different rhodium-based electron medium loadings; by comparing the reduction amount of NADH with different Rh dosages, it can be seen that with the increase of Rh, the reduction rate first increases and then decreases, indicating that too much Rh will produce enrichment and other phenomena, resulting in a decrease in yield. The optimal loading ratio is 0.6; Figure 5 The Fourier infrared peak images of the two photocatalysts COF and COF-Rh obtained in Comparative Example 1 and Example 1 are shown in Figure 1. From the peaks in the figure, it can be seen that after loading Rh, the peak intensity of H2O increases significantly, proving the successful loading of Rh on the COF structure. At the same time, the peak positions of C=C, C=O, and CN have not changed, which is also consistent with the previous XRD structure. The loading of Rh does not change the crystal form of the overall material. Figure 6 A comparison chart of the formic acid content per unit enzyme reduction of COF-Rh@FDH and COF-Rh plus free enzyme obtained in Example 2 and Implementation 1 is shown; as shown in the figure, COF-Rh@FDH and COF-Rh plus 3 mg of free enzyme are compared for formic acid reduction yields. From the data, it can be seen that the formic acid yield per unit enzyme of the loaded enzyme is about 9 times the formic acid yield per unit reduction of the free enzyme. At the same time, the loaded enzyme also has the performance of repeated use, which has dual advantages, and also proves the great advantage of the photocatalyst of the present invention in the application of photoenzyme cascade. Figure 7 The impedance comparison diagram of the two photocatalysts COF and COF-Rh obtained in Comparative Example 1 and Example 1; By comparing the impedance diagrams of COF-Rh and COF, it can be seen that the impedance radius of the photocatalyst is significantly reduced after loading the modified Rh, indicating that the modified photocatalytic material has a strong photogenerated electron performance. It also corresponds to the significant improvement in the ability to reduce NADH after loading Rh; Figure 8 The photocurrent response spectra of the two photocatalysts COF and COF-Rh obtained in Comparative Example 1 and Example 1 are shown; it can be seen from the figure that the photocurrent of COF-Rh is higher than that of COF, indicating that Rh loading can greatly increase the photogenerated current density of the catalyst, thereby improving its photocatalytic NADH regeneration activity.

Claims

1. A method for preparing a rhodium-based electron mediator and an enzyme co-loaded COF photocatalyst, comprising: Step 1) preparing a photocatalyst, denoted as COF, with 1,2,3-trialdehyde phloroglucinol as the donor and electron-deficient 2,2'-bipyridine-6,6'-diamine as the acceptor at a molar ratio of 1:2 between the donor and the acceptor; Step 2) mixing the COF obtained in step 1) with a methanol solution containing a certain amount of rhodium-based electron mediator [Rh(Cp*)C12]2, wherein the amount of [Rh(Cp*)C12]2 in the methanol solution is determined according to the mass ratio of the Rh content therein to the COF; stirring the mixed solution for 24 hours to modify the rhodium electron mediator into the COF, and the resulting product is a rhodium-loaded electron mediator COF photocatalyst, which is recorded as photocatalyst COF-Rh; Step 3) The COF-Rh prepared in step 2) and 5 mg of FDH were dissolved in 2 mL of phosphate buffer solution (PBS), reacted for 3 h, centrifuged and washed, and freeze-dried to obtain a COF framework material loaded with both electron mediators and enzymes, recorded as photocatalyst COF-Rh@FDH.

2. The preparation method according to claim 1, characterized in that: In the step 1), 0.0504 g of 1,2,3-trialdehyde phloroglucinol and 0.0671 g of 2,2'-bipyridine-6,6'-diamine are mixed in a molar ratio of 1:2 to obtain a mixture, and the mixture is added to 15 mL of solution A, wherein solution A is obtained by mixing o-dichlorobenzene and N,N-dimethylacetamide in a volume ratio of 1:

3. After ultrasonic mixing for 10 min, 5 mL of acetic acid solution (6 M) is added and ultrasonicated for 5 min. Then, the freezing-vacuuming-thawing process is repeated three times, and the mixture is reacted at 120°C for three days. After filtering and separation, the precipitate is washed with tetrahydrofuran, ultrapure water and methanol in sequence, and dried in a vacuum oven at 60°C to obtain the COF.

3. The preparation method according to claim 1, characterized in that: In the step 2), the COF prepared in step 1) and the methanol solution containing [Rh(Cp*)C12]2 are added into the reactor at a mass ratio of 0.4, 0.6, 0.8 and 1.0, and the reaction is carried out at a speed of 240 for 24 hours. After the reaction is completed, the solid is filtered and separated, and the solid is washed with ultrapure water and methanol in turn. After drying, the obtained product is the photocatalyst COF-Rh.

4. The preparation method according to claim 1, characterized in that: In the step 3), the COF-Rh and FDH enzymes prepared in step 2) are added to a PBS buffer solution according to a mass of 0.5, and reacted at room temperature for 3 h. After the reaction, the product is centrifuged, washed three times with a PBS solution, and then placed in a freeze dryer, freeze-dried for 24 h, and placed in a freezing layer for standby use, which is called COF-Rh@FDH.

5. A rhodium-based electron mediator and enzyme co-loaded COF material, which is obtained by the preparation method described in any one of claims 1 to 4.

6. The COF-Rh@FDH prepared by the present invention adopts a "two-in-one" strategy: the electron medium and FDH are co-anchored in the pores of the imine-linked COF to construct an integrated platform for heterogeneous CO2 photoconversion into formic acid. + , triethanolamine (TEOA) and PBS were added to the reaction system at a mass ratio of 2:1.45:20 and a volume ratio of 20:

1. The mixed solution of the above system was reacted for 30 min in the dark to achieve full mixing and adsorption. Then it was irradiated with a xenon lamp, a 420 nm filter was used, and samples were taken at intervals. After filtering with a 0.22 um filter head, formic acid products were detected by HPLC.

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