Biomimetic capsular core-shell type peroxidase coupling composite material, preparation method and application thereof

Abstract

The application discloses a biomimetic capsular core-shell type photoenzyme coupling composite material and a preparation method and application thereof, wherein the photoenzyme coupling composite material is self-assembled and grown on the surface of a ZIF-8 cavity with double-defect carbon nitride as a shell layer, and horseradish peroxidase (HRP) is confined in the ZIF-8 cavity; the preparation method is based on a "compartmentalization" strategy and an in-situ biomimetic mineralization method, wherein first, metal ligands of a ZIF-8 precursor are modified onto the double-defect carbon nitride through electrostatic interaction, then HRP is confined in the cavity of the ZIF-8 through in-situ biomimetic mineralization, and the double-defect carbon nitride is self-assembled and grown on the surface of the cavity of the ZIF-8.The biomimetic capsular core-shell type photoenzyme coupling composite material has both photocatalytic and enzymatic catalytic properties, and meanwhile, the coupling catalytic efficiency of the photoenzyme is improved, and the biomimetic capsular core-shell type photoenzyme coupling composite material can be used for in-situ production of H2O2 under light irradiation and catalytic degradation of organic pollutants through the action of H2O2 and HRP.

Description

Technical Field

[0001] This invention relates to photoenzyme-coupled composite materials, their preparation methods, and applications, specifically to biomimetic thylakoid-shell type photoenzyme-coupled composite materials, their preparation methods, and applications. Background Technology

[0002] The depletion of fossil fuels has triggered a series of energy and environmental problems. Reducing carbon emissions and alleviating dependence on non-renewable energy sources are crucial needs for the chemical manufacturing industry and the environmental sector. Enzyme catalysis, with its advantages of being green, mild, highly efficient, and selective, plays an important role in industrial production and technological research and development. However, enzyme-catalyzed reactions are limited in types and difficult to recycle, especially in the environmental field. Some oxidases require the addition of intermediate media such as NADH and H2O2 when treating wastewater. These intermediate media are costly and highly toxic, making it difficult to meet the needs of future sustainable green development and large-scale industrial production. Artificial photosynthetic catalysis systems are currently the most widely studied photocatalytic systems. Coupled with photocatalysts and light energy, they have become an effective strategy for generating reactive reaction intermediates under mild conditions, successfully achieving the synthesis of high-value chemicals, environmental treatment, and energy regeneration. However, the selectivity of photocatalytic reactions and the coordinated optimization and control of multiple processes remain challenging problems. Furthermore, this technology is still severely limited in driving the synthesis of complex organic molecules and treating high-concentration wastewater due to high costs.

[0003] Green plants in nature rely on their highly active catalytic components and highly coordinated catalytic processes to achieve efficient carbohydrate synthesis using solar energy and CO2, providing an ideal model system for artificial photosynthesis research and offering solutions to the increasingly severe energy and environmental crises. Inspired by the natural photosynthetic process, photoenzyme-coupled catalytic systems combining photocatalysis and enzyme catalysis are biomimetic model systems that simulate the natural photosynthetic process. Constructing high-performance photoenzyme-coupled artificial photosynthetic systems and exploring the coordination and optimization mechanisms among multiple (catalytic) processes can provide research models for exploring the mechanisms of natural photosynthesis, offer universal strategies for improving the efficiency and selective regulation of artificial photosynthesis, and overcome the limitations of natural enzyme catalysis. Currently, this has become one of the research hotspots in the fields of chemical synthesis, energy, and environmental science.

[0004] However, current photoenzyme-coupled catalytic systems still focus on the development of highly active catalysts, lacking in-depth exploration of the compatibility of various catalytic processes within the system and the efficient conversion between energy flow and mass flow. This results in three main key scientific problems for photoenzyme-coupled catalytic systems: 1) the compatibility between photogenerated holes and their derived radicals and enzyme catalysts; 2) the rapid transfer of photogenerated electrons between photogenerated electrons and enzyme catalysts; and 3) the influence of light on the structure and activity of enzyme catalysts. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a biomimetic thylakoid-shell type photoenzyme coupling composite material with good compatibility between photocatalyst and enzyme catalyst, high catalytic efficiency and good stability. Furthermore, this invention also provides a method for preparing the photoenzyme coupling composite material and its application.

[0006] Technical solution: The biomimetic thylakoid-shell type photoenzyme coupling composite material of the present invention is self-assembled and grown on the surface of ZIF-8 cavity with double-defect carbon nitride as the shell, and horseradish peroxidase is confined in ZIF-8 cavity.

[0007] Furthermore, the dual-defect carbon nitride is acetylacetone-modified carbon nitride.

[0008] The aforementioned biomimetic vesicle-shell type photoenzyme coupling composite material is prepared using an in-situ biomimetic mineralization method based on a "compartmentalization" strategy. The preparation method includes the following steps:

[0009] (1) First, disperse the double-defect carbon nitride in a solvent, then add the precursor metal ligand zinc nitrate hexahydrate of ZIF-8, and then sonicate to obtain solution A;

[0010] (2) Mix the ZIF-8 precursor organic ligand 2-methylimidazole, horseradish peroxidase and polyvinylpyrrolidone thoroughly in a solvent to obtain solution B;

[0011] (3) Add solution A to solution B and stir the mixture thoroughly to obtain a mixture. After centrifugation, washing with water and freeze-drying, the biomimetic thylakoid-shell type photoenzyme coupling composite material is obtained.

[0012] Further, in step (1), the double-defect carbon nitride is acetylacetone-modified carbon nitride, and the preparation method is as follows:

[0013] (11) Dissolve acetylacetone and urea in isopropanol and sonicate. Then dry the resulting mixture to obtain white crystals.

[0014] (12) The white crystals were calcined to obtain acetylacetone-modified carbon nitride.

[0015] Further, the molar ratio of acetylacetone to urea is 0.025-0.2:1; the ratio of the amount of acetylacetone, urea and isopropanol is 0.25-2.0 mL: 6 g: 25 mL; and the calcination conditions are: calcination at 540-560℃ for 2-2.5 h.

[0016] Further, in step (1), the mass ratio of the double-defect carbon nitride to zinc nitrate hexahydrate is 1:24.8-10:24.8, preferably 1:14.9, the concentration of the double-defect carbon nitride in solution A is 3-10 mg / mL, preferably 5 mg / mL, and the ultrasonic treatment time is 0.5-1 h.

[0017] Further, in step (2), the mass ratio of 2-methylimidazole, horseradish peroxidase and polyvinylpyrrolidone is 16.4:0.4:2-16.4:1.2:2; the concentration of horseradish peroxidase in solution B is 0.4-1.2 mg / mL.

[0018] Further, in step (3), the volume ratio of solution A to solution B is 1:5-1:10, the concentration ratio of zinc nitrate hexahydrate in solution A to 2-methylimidazole in solution B is 1:4-1:8, and the mass ratio of double-defect carbon nitride in solution A to horseradish peroxidase in solution B is 0.6-2.5:1, preferably 1:1.

[0019] Furthermore, in step (3), the stirring reaction time is 30-120 min.

[0020] In the entire preparation process, this invention first utilizes acetylacetone-modified dual-defect carbon nitride as a photocatalyst, with the defect types being C doping and surface -OH functional group modification. The Zn in the precursor metal ligand of ZIF-8 is then catalyzed by electrostatic interactions generated by ultrasound and van der Waals forces. 2+ Uniformly grown onto the surface of double-defect carbon nitride, a mixture of 2-methylimidazole (2-MeIM), the organic ligand of the ZIF-8 precursor, horseradish peroxidase (HRP), and polyvinylpyrrolidone (PVP) was then added. Through in-situ biomimetic mineralization, 2-methylimidazole enriched around the PRP-modified horseradish peroxidase, along with Zn on the double-defect carbon nitride, was deposited. 2+ Rapid nucleation occurs, during which dual-defect carbon nitride grows on the surface of ZIF-8 along with its nucleation, forming a shell; ZIF-8 acts as a compartment, confining horseradish peroxidase within it, ultimately yielding a core-shell photoenzyme coupling composite material.

[0021] The above-mentioned biomimetic vesicle-shell type photoenzyme coupling composite material is used as a catalyst in the in-situ production of H2O2 and degradation of organic pollutants.

[0022] Furthermore, the organic pollutants include phenolic and amine pollutants.

[0023] Furthermore, the principle of in-situ H2O2 production and catalytic degradation of organic pollutants is as follows: Under visible light irradiation, the photocatalyst double-defect carbon nitride in the biomimetic thylakoid core-shell photoenzyme coupling composite material generates photogenerated electrons to reduce O2 to H2O2. Horseradish peroxidase uses H2O2 in situ to catalyze the degradation of phenols and amines, while ZIF-8 protects the enzyme from the effects of light and photoderived free radicals, thereby achieving highly efficient photoenzyme coupling catalytic performance.

[0024] Invention Principle: Inspired by the structure and function of thylakoids in natural photosynthetic systems, this invention explores a new pathway for constructing artificial photosynthetic systems based on photoenzyme coupling catalysis. Utilizing the "confining effect" and "compartmentalization effect," a biomimetic thylakoid core-shell photoenzyme coupling composite material (HZA) is designed and prepared for coupling photoenzyme catalysis processes, achieving efficient substrate conversion. In this invention, the preparation method of the biomimetic thylakoid core-shell photoenzyme coupling composite material is based on an in-situ biomimetic mineralization method using a "compartmentalization" strategy. The precursor metal ligand of ZIF-8 is modified with a double-defect carbon nitride through electrostatic interactions to obtain solution A. Then, HRP and PVP are doped into the organic ligand of the ZIF-8 precursor to obtain solution B. Finally, solution A is added dropwise to solution B, and in-situ biomimetic mineralization confines HRP within the cavity of ZIF-8. In this invention, a biomimetic thylakoid-shell type photoenzyme coupling system is constructed based on a "compartmentalization" strategy. ZIF-8 acts as a "compartment," confining the photocatalytic and enzymatic reactions to the outside and inside of the "compartment," respectively. This system possesses both photocatalytic and enzymatic properties, simultaneously improving the catalytic efficiency of the photoenzyme coupling. It can be used to in-situ convert O2 to 2e under light irradiation. - The oxygen reduction pathway generates H2O2, which then reacts with HRP to catalyze the degradation of organic pollutants. The biomimetic thylakoid-shell photoenzyme coupling catalyst requires no sacrificial agents, avoiding the waste of photogenerated holes and ensuring full utilization of photogenerated carriers. Its compartmentalized structure prevents the influence of photocatalyst-generated active species and light on the enzyme's structure and activity. The catalyst's preparation process is simple, green, and feasible, using readily available and inexpensive raw materials, and has wide applications, aligning with the concept of sustainable green development and the "dual-carbon" strategy.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0026] (1) This invention constructs a new method for a photoenzyme coupled artificial photosynthesis system based on the "compartmentalization" strategy. The "compartmentalization" improves the compatibility between the photocatalyst and the enzyme catalyst, and can simultaneously achieve efficient in-situ regeneration of H2O2 by photocatalysis and efficient coupling between the photocatalyst and the enzyme catalyst under light irradiation, which significantly improves the removal rate of phenols and amines.

[0027] (2) The preparation process of this invention is green and environmentally friendly, and the raw material cost is low, which meets the requirements of sustainable green development. Attached Figure Description

[0028] Figure 1 SEM images of the biomimetic thylakoid-shell type photoenzyme coupling composite materials prepared in Examples 1-5;

[0029] Figure 2 The UV-vis DRS spectra, transient photocurrent (it), and electrochemical impedance (EIS) plots of ZA in Comparative Example 1 and HZA in Example 2 are shown.

[0030] Figure 3 This is a reaction process diagram of in-situ H2O2 production from ZA in Comparative Example 1 and HZA in Example 2, as measured in Example 6;

[0031] Figure 4 The graph shows the first-order kinetic fit values ​​of ABTS oxidation for ZA in Comparative Example 1 and HZA in Example 2, measured under darkness and light conditions in Example 7.

[0032] Figure 5 This is a graph showing the removal rates of ZA in Comparative Example 1 and HZA in Example 2 for different substrates, as measured in Example 8.

[0033] Figure 6 This is a stability diagram of HZA and HA in Example 2, measured in Example 9, under light and high temperature treatment. Detailed Implementation

[0034] The present invention will now be further described in conjunction with specific embodiments and accompanying drawings.

[0035] I. Terminology

[0036] In this invention, "O2" refers to oxygen, "H2O2" refers to hydrogen peroxide, "CO2" refers to carbon dioxide, "ZIF-8" refers to zeolite imidazole framework material, "PVP" refers to polyvinylpyrrolidone, "HRP" refers to horseradish peroxidase, "Zn(NO3)2·6H2O" refers to zinc nitrate hexahydrate, "2-MeIM" refers to 2-methylimidazole, "NAPDH" refers to reduced coenzyme, "BPA" refers to bisphenol A, "2,4-DCP" refers to 2,4-dichlorophenol, "AN" refers to aniline, and "ABTS" refers to 2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid.

[0037] Unless otherwise specified or limited, the term "water" in this invention refers to deionized water or ultrapure water.

[0038] II. Implementation Plan

[0039] When peroxidases catalyze the conversion of substrates, redox equivalents (such as H2O2) are involved. High concentrations of H2O2 can lead to enzyme inactivation, and free enzymes are not conducive to recovery and reuse. In photoenzyme-coupled catalytic systems, light and photoderived free radicals can damage the enzyme structure, resulting in reduced enzyme catalytic performance. Based on these challenges, this invention proposes a biomimetic thylakoid core-shell type HZA photoenzyme-coupled catalytic system based on a "compartmentalization" strategy. This system can simultaneously achieve in-situ photocatalytic H2O2 generation, compatibility between light and photocatalysts and enzyme catalysts, and improved photoenzyme-coupled catalytic conversion performance of substrates.

[0040] To achieve the above technical solution, this invention provides a biomimetic thylakoid-shell type HZA photoenzyme coupling catalyst. Unless otherwise specified, the experimental methods described below are standard laboratory methods. Unless otherwise specified, the experimental materials described below are commercially available.

[0041] In this invention, the OD value can be measured using an ultraviolet spectrophotometer (UV 2700) and an enzyme-linked immunosorbent assay (ELISA) reader to evaluate the material's H2O2 production performance and enzyme activity.

[0042] In this invention, the synthesized material can be characterized by scanning electron microscopy (SEM), proving that the core-shell composite material was successfully synthesized.

[0043] In this invention, the light absorption range and electron transfer rate of a material can be characterized by solid-state ultraviolet diffuse reflectance (UV-vis DRS) spectra and transient photocurrent (it) curves, demonstrating the material's ability to absorb visible light and its interface electron transfer efficiency.

[0044] Example 1: The biomimetic thylakoid-shell photoenzyme coupling composite material (hereinafter referred to as HZA) provided in this example uses double-defect carbon nitride as the shell, which is self-assembled and grown outside the cavity of ZIF-8. Horseradish peroxidase is confined inside the cavity of ZIF-8. ZIF-8 acts as a compartment, separating the photocatalyst and the enzyme catalyst. The double-defect carbon nitride (hereinafter referred to as ACN) is acetylacetone-modified carbon nitride.

[0045] The above-mentioned photoenzyme coupling composite material is prepared using a one-pot in-situ biomimetic mineralization method, and the steps are as follows:

[0046] (1) Preparation of ACN: Dissolve 0.5 mL of acetylacetone and 6 g of urea in 25 mL of isopropanol and sonicate. Then dry the resulting mixture to obtain white crystals. Place the white crystals in a crucible and calcine at 550 °C for 1 h to obtain ACN.

[0047] (2) Accurately weigh 15 mg ACN into a 10 mL centrifuge tube, add 5 mL of ultrapure water, and sonicate for 10 min to disperse it evenly; add 371.3 mg of accurately weighed Zn(NO3)2·6H2O, sonicate for 1 h, and record the mixture as solution A.

[0048] (3) Accurately weigh 410 mg 2-MeIM into a 50 mL beaker, add 25 mL of ultrapure water, shake well, then add 50 mg PVP and 25 mg HRP that have been accurately weighed, and continue to stir magnetically to make it evenly mixed. Record the mixture as solution B.

[0049] (3) Then, the solution A obtained above was added dropwise to solution B, and magnetic stirring was continued; after stirring for 1 hour, centrifugation was performed at 10,000 r / min, and the supernatant was removed; then the precipitate was washed with ultrapure water to remove unreacted raw materials; finally, the obtained sample was vacuum dried for 24 hours to obtain sample HZA. 0.6:1 (ACN:HRP mass ratio is 0.6:1).

[0050] Example 2: The biomimetic thylakoid-shell photoenzyme coupling composite material provided in this example uses double-defect carbon nitride as the shell, which is self-assembled and grown outside the cavity of ZIF-8. Horseradish peroxidase is confined inside the cavity of ZIF-8. ZIF-8 acts as a compartment to separate the photocatalyst and the enzyme catalyst. The double-defect carbon nitride (hereinafter referred to as ACN) is acetylacetone-modified carbon nitride.

[0051] The above-mentioned photoenzyme coupling composite material is prepared using a one-pot in-situ biomimetic mineralization method, and the steps are as follows:

[0052] (1) Accurately weigh 25 mg ACN into a 10 mL centrifuge tube, add 5 mL of ultrapure water, and sonicate for 10 min to disperse it evenly; add 371.3 mg Zn(NO3)2·6H2O accurately weighed into it, sonicate for 1 h, and record the mixture as solution A.

[0053] (2) Accurately weigh 410 mg of 2-MeIM into a 50 mL beaker, add 25 mL of ultrapure water, shake well, then add 50 mg of accurately weighed PVP and 25 mg of HRP, continue to stir magnetically to mix evenly, and record the mixture as solution B.

[0054] (3) Then, the solution A obtained above was added dropwise to solution B, and magnetic stirring was continued; after stirring for 1 hour, centrifugation was performed at 10,000 r / min, and the supernatant was removed; then the precipitate was washed with ultrapure water to remove unreacted raw materials; finally, the obtained sample was vacuum dried for 24 hours to obtain sample HZA. 1:1 (ACN:HRP mass ratio is 1:1).

[0055] Example 3: The biomimetic thylakoid-shell photoenzyme coupling composite material provided in this example uses double-defect carbon nitride as the shell, which is self-assembled and grown outside the cavity of ZIF-8. Horseradish peroxidase is confined inside the cavity of ZIF-8. ZIF-8 acts as a compartment to separate the photocatalyst and the enzyme catalyst. The double-defect carbon nitride (hereinafter referred to as ACN) is acetylacetone-modified carbon nitride.

[0056] The above-mentioned photoenzyme coupling composite material is prepared using a one-pot in-situ biomimetic mineralization method, and the steps are as follows:

[0057] (1) Accurately weigh 50 mg ACN into a 10 mL centrifuge tube, add 5 mL of ultrapure water, and sonicate for 10 min to disperse it evenly; add 371.3 mg Zn(NO3)2·6H2O that has been accurately weighed, sonicate for 1 h, and record the mixture as solution A.

[0058] (2) Accurately weigh 410 mg of 2-MeIM into a 50 mL beaker, add 25 mL of ultrapure water, shake well, then add 50 mg of accurately weighed PVP and 25 mg of HRP, continue to stir magnetically to mix evenly, and record the mixture as solution B.

[0059] (3) Then, the solution A obtained above was added dropwise to solution B, and magnetic stirring was continued; after stirring for 1 hour, centrifugation was performed at 10,000 r / min, and the supernatant was removed; then the precipitate was washed with ultrapure water to remove unreacted raw materials; finally, the obtained sample was vacuum dried for 24 hours to obtain sample HZA. 2:1 (ACN:HRP mass ratio is 2:1).

[0060] Example 4: The biomimetic thylakoid-shell type photoenzyme coupling composite material provided in this example uses double-defect carbon nitride as the shell layer, which is self-assembled and grown outside the cavity of ZIF-8. Horseradish peroxidase is confined inside the cavity of ZIF-8. ZIF-8 acts as a compartment to separate the photocatalyst and the enzyme catalyst. The double-defect carbon nitride (hereinafter referred to as ACN) is acetylacetone-modified carbon nitride.

[0061] The above-mentioned photoenzyme coupling composite material is prepared using a one-pot in-situ biomimetic mineralization method, and the steps are as follows:

[0062] (1) Accurately weigh 25 mg ACN into a 10 mL centrifuge tube, add 5 mL of ultrapure water, and sonicate for 10 min to disperse it evenly; add 371.3 Zn(NO3)2·6H2O accurately, sonicate for 1 h, and record the mixture as solution A.

[0063] (2) Accurately weigh 410 mg of 2-MeIM into a 50 mL beaker, add 25 mL of ultrapure water, shake well, then add 50 mg of accurately weighed PVP and 10 mg of HRP, continue to stir magnetically to mix evenly, and record the mixture as solution B.

[0064] (3) Then, the solution A obtained above was added dropwise to solution B, and magnetic stirring was continued; after stirring for 1 hour, centrifugation was performed at 10,000 r / min, and the supernatant was removed; then the precipitate was washed with ultrapure water to remove unreacted raw materials; finally, the obtained sample was vacuum dried for 24 hours to obtain sample HZA. 2.5:1 (ACN:HRP mass ratio is 2.5:1).

[0065] Example 5: The biomimetic thylakoid-shell type photoenzyme coupling composite material provided in this example uses double-defect carbon nitride as the shell layer, which is self-assembled and grown outside the cavity of ZIF-8. Horseradish peroxidase is confined inside the cavity of ZIF-8. ZIF-8 acts as a compartment to separate the photocatalyst and the enzyme catalyst. The double-defect carbon nitride (hereinafter referred to as ACN) is acetylacetone-modified carbon nitride.

[0066] The above-mentioned photoenzyme coupling composite material is prepared using a one-pot in-situ biomimetic mineralization method, and the steps are as follows:

[0067] (1) Accurately weigh 25 mg ACN into a 10 mL centrifuge tube, add 5 mL of ultrapure water, and sonicate for 10 min to disperse it evenly; add 371.3 mg Zn(NO3)2·6H2O accurately weighed into it, sonicate for 1 h, and record the mixture as solution A.

[0068] (2) Accurately weigh 410 mg of 2-MeIM into a 50 mL beaker, add 25 mL of ultrapure water, shake well, then add 50 mg of accurately weighed PVP and 30 mg of HRP, continue to stir magnetically to mix evenly, and record the mixture as solution B.

[0069] (3) Then, the solution A obtained above was added dropwise to solution B, and magnetic stirring was continued; after stirring for 1 hour, centrifugation was performed at 10,000 r / min, and the supernatant was removed; then the precipitate was washed with ultrapure water to remove unreacted raw materials; finally, the obtained sample was vacuum dried for 24 hours to obtain sample HZA. 0.83:1 (ACN:HRP mass ratio is 0.83:1).

[0070] Comparative Example 1: The ZA composite material provided in this comparative example uses double-defect carbon nitride as a shell, which is self-assembled and grown outside the cavity of ZIF-8. The double-defect carbon nitride (hereinafter referred to as ACN) is acetylacetone-modified carbon nitride.

[0071] The above-mentioned ZA composite material is prepared using a one-pot method, and the steps are as follows:

[0072] (1) Accurately weigh 25 mg ACN into a 10 mL centrifuge tube, add 5 mL of ultrapure water, and sonicate for 10 min to disperse it evenly; add 371.3 mg Zn(NO3)2·6H2O accurately weighed into it, sonicate for 1 h, and record the mixture as solution A.

[0073] (2) Accurately weigh 410 mg of 2-MeIM into a 50 mL beaker, add 25 mL of ultrapure water, and stir magnetically to mix it evenly. Record the mixture as solution B.

[0074] (3) Then the solution A obtained above is added dropwise to solution B and magnetic stirring is continued; after stirring for 1 hour, centrifugation is performed at 10,000 r / min and the supernatant is removed; then the precipitate is washed with ultrapure water to remove unreacted raw materials; finally, the obtained sample is vacuum dried for 24 hours to obtain sample ZA composite material.

[0075] HZA synthesized in Examples 1-5 with different ACN to HRP concentration ratios were characterized by SEM. Figure 1 As shown, from Figure 1 The SEM images (ae) show that the morphology of HZA varies considerably depending on the ratio of photocatalyst to enzyme concentration. When the concentration ratio of ACN to HRP is 1:1, the HZA exhibits a dodecahedral structure with surface wrinkles and is uniformly dispersed. TEM characterization of the HZA with an ACN:HRP ratio of 1:1 was performed to further illustrate its morphology. Figure 1 As can be seen in (f), there is a clear difference in brightness between the edge and the interior of the wrinkled dodecahedron, and it can be clearly seen that the edge of the dodecahedron is assembled from ultrathin carbon nanosheets, which proves the successful synthesis of the HZA core-shell structure. This assembly method can generate a variety of nanopores, which is beneficial to the transport and separation of charge carriers and the easy transport of substrates into the interior of the catalyst.

[0076] Figure 2 The UV-vis DRS spectra (a), transient photocurrent (it) curves (b), and electrochemical impedance (EIS) plots (c) of ZA prepared in Comparative Example 1 and HZA prepared in Example 2 are shown below. Figure 2 As can be seen in (a), both ZA and HZA have wide visible light absorption ranges. Compared to ZA, HZA's visible light absorption range shows a significant red shift, indicating its higher solar energy utilization efficiency. Figure 2 As can be seen from (b) and (c), HZA has a high transient photocurrent and a small electrochemical impedance, indicating that HZA has excellent electron transfer efficiency and easy separation of interface charges.

[0077] Example 6: Application of biomimetic thylakoid-shell type photoenzyme coupling composite material in in-situ H2O2 production, the steps are as follows:

[0078] In ultrapure water, 0.2 mg / mL of ZA prepared in Comparative Example 1 and HZA prepared in Example 2 were added, with a total reaction volume of 50 mL. The reaction was first carried out in the dark for 1 hour, and then irradiated under a 450 nm LED lamp (light intensity of 100 mW / cm²). 2 The reaction proceeded for 1 hour, with samples taken every 10 minutes and filtered through a 0.22 μm filter. The absorbance at OD551 was measured using a UV spectrophotometer (UV 2700). The reaction process of H2O2 was as follows: Figure 3 As shown, in HZA, HRP utilizes the in-situ generated H2O2, resulting in a lower H2O2 yield compared to ZA, which also demonstrates the photoenzyme coupling performance.

[0079] Example 7: Enzyme activity determination of photoenzyme coupling composite material under dark / light conditions

[0080] Enzyme activity assay of the photoenzyme coupling material under dark conditions: 10 μL of ZA prepared in Comparative Example 1 (1 mg / mL) and HZA prepared in Example 2 were added to a 0.1 M phosphate buffer solution at pH 6.5. 40 μL of ABTS solution (1.5 mM) and 50 μL of H2O2 (0.1 M) were also added, for a total reaction volume of 300 μL. The mixture was incubated at 25 °C for 1 min, and the absorbance at OD420 was measured over time using a microplate reader. The first-order fitted value of the ABTS reaction catalyzed by the material under dark conditions (k) was also determined. obs )like Figure 4 As shown. In the dark, ZA has almost no catalytic performance for ABTS, k obs =0.007min -1 HZA has catalytic properties for ABTS, k obs =0.024min -1 It is 3.4 times that of ZA.

[0081] Enzyme activity assay of the photoenzyme coupling material under illumination: 1 mg / mL of ZA prepared in Comparative Example 1 and HZA prepared in Example 2 were added to a 0.1 M phosphate buffer solution at pH 6.5, followed by 100 μL of 1.5 mM ABTS solution, for a total reaction volume of 10 mL. The mixture was ultrasonically dispersed at 25 °C. The reaction was then carried out under a 450 nm LED (100 mW / cm²). 2 Irradiated under a light source for 10 minutes, with samples taken every 2 minutes to measure the absorbance at OD420. The first-order fitted value of the reaction kinetics of the material catalyzing ABTS under illumination (k...) obs )like Figure 4As shown. Under illumination, ZA enhances the catalytic performance of ABTS, k obs =0.016min -1 HZA significantly enhances the catalytic performance of ABTS, k obs =0.074min -1 It is 4.6 times that of ZA. By comparing the activity of HZA under light and dark conditions, the results showed that HZA(k) activity under light was significantly higher than that under light conditions. obs =0.074min -1 The activity of HZA(k) is under dark conditions. obs =0.024min -1 The rate was 3.2 times that of the enzyme, proving that photoenzyme coupling promoted the rate of enzyme-catalyzed reaction.

[0082] Example 8: Catalytic degradation of organic pollutants by biomimetic thylakoid-shell type photoenzyme coupled composite material

[0083] In 50 mL of 10 mg / L aqueous solutions of different substrates (including BPA, 2,4-DCP, and AN), 10 mg of ZA prepared in Comparative Example 1 or HZA prepared in Example 2 was added as a catalyst and ultrasonically dispersed. The mixture was then magnetically stirred for 1 h in a dark environment at 25 °C, followed by reaction under 450 nm LED (100 mW / cm²). 2 Under light irradiation, the reaction was carried out at 25℃ for 1 hour. 1 mL of sample was taken every 10 minutes and filtered through a 0.22 μm filter. The amount of substrate was detected by ultra-high phase liquid chromatography (HPLC), and the removal rate of the substrate by the material was calculated using Formula 1.

[0084] Figure 5 The results show that HZA exhibits excellent photoenzyme coupling catalytic performance, with removal rates of BPA (99.1%), 2,4-DCP (88.5%), and AN (60.3%), respectively, while the removal rates of ZA for the substrates are only BPA (55.6%), 2,4-DCP (77.7%), and AN (34.7%), respectively.

[0085] Removal rate = (initial concentration - concentration at time t) / initial concentration × 100% (Formula 1)

[0086] Example 9: Comparison between the biomimetic thylakoid-shell photoenzyme coupling composite material system and the ordinary HA photoenzyme material system

[0087] To demonstrate the protective effect of the "compartmentalization" strategy on the enzyme in the biomimetic thylakoid-shell type HZA photoenzyme coupling system, HRP enzyme was directly adsorbed onto the photocatalyst ACN to obtain the HA photoenzyme coupling system (where the mass ratio of ACN to HRP is 1:1).

[0088] The enzyme activities of the two were then compared after 1 hour of light exposure and 1 hour of high-temperature treatment. 10 mg of HZA and HA prepared in Example 2 were dispersed in 10 mL of ultrapure water and ultrasonically dispersed evenly. The enzyme activity in the measured samples was expressed as relative activity, that is, the ratio of the initial reaction rate of the sample after treatment to the initial reaction rate before treatment.

[0089] Enzyme activity stability under light conditions: HZA and HA were tested at 450 nm and 100 mW / cm². 2 The samples were irradiated under an LED lamp for 1 hour to test their catalytic activity. Each reaction was performed in triplicate, and the average value was taken.

[0090] Enzyme activity stability under high temperature conditions: HZA and HA were placed at 70℃ and 90℃ for 1 h, respectively. After the samples cooled to room temperature, the catalytic activity of the samples was tested. Each reaction was performed in triplicate, and the average value was taken.

[0091] Figure 6 The stability of HZA and HA under light and high temperature was investigated. The results showed that the enzyme activity of uncompartmented HA decreased by 83.4% after 1 hour of light exposure, while that of compartmentalized HZA decreased by only 36.9%. Similarly, under treatment at 70℃ and 90℃, the enzyme activity of HZA decreased by only 23.3% and 36.1%, respectively, while the enzyme activity of HA decreased significantly by 53.8% and 63.2%. These results indicate that in the biomimetic thylakoid core-shell type HZA photoenzyme coupling composite material system, porous ZIF-8 effectively separates photocatalysis and enzyme catalysis as "compartments," giving the HZA system good compatibility and connectivity.

Claims

1. A biomimetic thylakoid-shell type photoenzyme coupling composite material, characterized in that, Horseradish peroxidase is confined within the ZIF-8 cavity by self-assembly of dual-defect carbon nitride as the shell. The defect types of the dual-defect carbon nitride are C doping and surface -OH functional group modification. The preparation method of the biomimetic thylakoid-shell type photoenzyme coupling composite material includes the following steps: (1) First, disperse the double-defect carbon nitride in a solvent, then add the precursor metal ligand zinc nitrate hexahydrate of ZIF-8, and sonicate to obtain solution A; the mass ratio of the double-defect carbon nitride to zinc nitrate hexahydrate is 1:7.4-1:24.8, and the concentration of the double-defect carbon nitride in solution A is 1.5-10 mg / mL; (2) The precursor organic ligand 2-methylimidazole, horseradish peroxidase and polyvinylpyrrolidone of ZIF-8 are thoroughly mixed in a solvent to obtain solution B; the mass ratio of 2-methylimidazole, horseradish peroxidase and polyvinylpyrrolidone is 16.4:0.4:2-16.4:1.2:2; the concentration of horseradish peroxidase in solution B is 0.4-1.2 mg / mL; (3) Add solution A dropwise to solution B, stir thoroughly to obtain a mixture, and then centrifuge, wash with water and freeze dry the mixture to obtain the biomimetic thylakoid-shell type photoenzyme coupling composite material; the volume ratio of solution A to solution B is 1:5-1:10, the concentration ratio of zinc nitrate hexahydrate in solution A to 2-methylimidazole in solution B is 1:4-1:8, and the mass ratio of double-defect carbon nitride in solution A to horseradish peroxidase in solution B is 0.6-2.5:

1.

2. The biomimetic thylakoid-shell type photoenzyme coupling composite material according to claim 1, characterized in that, In step (1), the double-defect carbon nitride is acetylacetone-modified carbon nitride, and the preparation method is as follows: (11) Acetylacetone and urea were dissolved in isopropanol and ultrasonically treated. The resulting mixture was dried to obtain white crystals. (12) The white crystals were calcined to obtain carbon nitride modified with acetylacetone.

3. The biomimetic thylakoid-shell type photoenzyme coupling composite material according to claim 2, characterized in that, The molar ratio of acetylacetone to urea is 0.025-0.2:1; the ratio of the amount of acetylacetone, urea and isopropanol is 0.25-2.0 mL: 6 g: 25 mL; the calcination conditions are: calcination at 540-560℃ for 2-2.5 h.

4. The biomimetic thylakoid-shell type photoenzyme coupling composite material according to claim 1, characterized in that, In step (1), the ultrasonic treatment time is 0.5-1 h.

5. The biomimetic thylakoid-shell type photoenzyme coupling composite material according to claim 1, characterized in that, In step (3), the stirring reaction time is 30-120 min.

6. The application of the biomimetic thylakoid-shell type photoenzyme coupling composite material as described in claim 1 as a catalyst in in-situ H2O2 production and organic pollutant degradation.

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