Mof enzyme catalytic material with light regulation characteristics and preparation method thereof

Abstract

The application relates to the technical field of enzyme catalysis, and discloses a MOF enzyme catalysis material with light regulation characteristics and a preparation method thereof, which comprises the following steps: mixing an ammonia polymer, triethylamine, an organic solvent, an acyl chloride solution and propionyl chloride to perform an amidation reaction to obtain a light response polymer; and mixing the light response polymer with a functional enzyme, water, a solution of an organic ligand, and a solution of a metal salt to perform a mixing reaction to obtain the MOF enzyme catalysis material with the light regulation characteristics. The application provides a light response enzyme catalysis composite material with high stability, intelligent controllability, easy recovery and strong universality and a preparation method thereof. The material utilizes the synergistic effect of a MOF rigid framework and a flexible polymer to construct a rigid-flexible combined multiple regulation system, so that the activity regulation of an enzyme in a confined environment is realized. The material has wide application prospects in the fields of intelligent biosensing, precision medicine, environmental governance and green synthesis.

Description

Technical Field

[0001] This invention relates to the field of enzyme catalysis technology, and more specifically, to a MOF enzyme catalytic material with photomodulation properties and its preparation method. Background Technology

[0002] Enzymes are biological macromolecules with catalytic functions produced by living cells. As a typical class of biocatalysts, they have outstanding advantages over traditional chemical catalysts, such as high catalytic efficiency, strong substrate specificity, mild reaction conditions, and green and pollution-free properties, and have shown great potential in fields such as chemical engineering, medicine, food, and environmental remediation.

[0003] Despite the significant advantages of enzyme catalysis technology, the inherent defects of naturally occurring free enzymes severely restrict their large-scale, industrial application. In actual catalytic reaction systems, free enzymes are completely exposed to the reaction environment, lacking effective protection of their molecular structure. They are susceptible to the influence of external reaction conditions such as temperature, pH, organic solvents, and high salt concentrations, leading to conformational collapse, inactivation of active sites, and protein denaturation. This results in rapid decline in catalytic activity and a significantly shortened lifespan. Furthermore, the uniform dispersion of free enzymes in the reaction system makes separation and recovery after the reaction difficult, hindering recycling. This not only significantly increases the raw material costs and energy consumption of industrial catalysis but also causes problems such as enzyme residues in the product system, difficulty in product purification, and insufficient product purity. These issues severely limit the promotion and application of enzyme catalysis technology in continuous, large-scale industrial production.

[0004] To effectively overcome the aforementioned defects of free enzymes, immobilized enzyme technology has become a mainstream research direction in the field of biocatalysis. This technology loads enzyme molecules onto the surface or internal pores of a carrier material through physical adsorption, embedding, covalent bonding, and coordination immobilization. This provides stable spatial protection for enzyme molecules, effectively maintaining their spatial conformation, enhancing their environmental tolerance and structural stability. It also enables rapid enzyme separation and reuse, significantly reducing production costs, simplifying processes, and improving product purity. Among numerous enzyme immobilization carrier materials, metal-organic frameworks (MOFs) possess unique advantages such as large specific surface area, controllable pore structure, uniform pore size, abundant surface functional groups, and good biocompatibility. Compared to traditional porous carbon, molecular sieves, and polymer gels, MOFs can achieve efficient loading and precise immobilization of enzyme molecules, maximizing the preservation of the enzyme's inherent catalytic activity. This makes MOFs the preferred material for immobilized enzyme carriers and provides an effective technical path to overcome the bottlenecks in the industrial application of free enzymes.

[0005] Currently, most existing MOF-based enzyme immobilization technologies utilize structurally stable rigid porous MOFs as supports. These rigid MOFs possess excellent structural and chemical stability, effectively preventing the collapse and degradation of the support itself in the reaction system, and providing basic structural protection for the immobilized enzyme. However, the rigid support's framework structure is fixed, and its spatial configuration cannot be dynamically adjusted, representing a typical static immobilization mode with significant technical limitations. In the dynamic process of enzyme-catalyzed reactions, the entire process of substrate binding, catalytic reaction, and product desorption requires dynamic fine-tuning of the spatial conformation of the enzyme's active site to adapt to the reaction progress. Rigid MOF supports cannot dynamically intervene in and precisely regulate the spatial structure and microenvironment characteristics of the immobilized enzyme's active site in response to the dynamic changes in the catalytic reaction.

[0006] This static immobilization mode can only achieve simple loading and passive protection of enzyme molecules, and cannot adapt to the dynamic regulation requirements of enzyme catalytic reactions. It is difficult to achieve precise control over reaction rate, reaction selectivity, and product-directed generation, and it is prone to problems such as restricted substrate diffusion, incomplete catalytic reaction, by-product formation, and low efficiency of product-directed synthesis. It cannot meet the stringent industrial requirements of high-end biocatalysis for controllable reaction process, precise product orientation, and adjustable catalytic efficiency. With the rapid development of intelligent biocatalysis and precision biomanufacturing technologies, the demand for intelligent and controllable regulation of immobilized enzyme systems is becoming increasingly urgent. In-situ and real-time dynamic regulation of enzyme activity has become the core key to upgrading biocatalysis technology and breaking through the barriers to high-end industrial applications. Therefore, developing novel MOF support materials with dynamic response and in-situ precise regulation of activity, breaking through the limitations of traditional static immobilization technology, is a technical challenge that urgently needs to be solved in the fields of immobilized enzymes and intelligent biocatalysis. Summary of the Invention

[0007] In view of this, the present invention proposes a MOF enzyme catalytic material with photomodulation properties and its preparation method, aiming to solve the problem that existing immobilized enzyme technology cannot precisely control the reaction process.

[0008] This invention proposes a method for preparing MOF enzyme catalytic materials with photomodulation properties, which includes the following steps: 1) An amidation reaction was carried out by mixing an ammonia polymer, triethylamine, an organic solvent, an acyl chloride solution, and propionyl chloride to obtain a photoresponsive polymer; 2) A MOF enzyme catalytic material with photo-regulated properties is obtained by mixing and reacting photoresponsive polymers with solutions of functional enzymes, water, organic ligands, and metal salts. The solute in the acyl chloride solution is an azophenylformyl chloride compound.

[0009] Preferably, the ammonia polymer includes one or more of branched polyethyleneimine, polyallylamine, polyacrylamide, and hyperbranched polyetheramine; The solvent in the acyl chloride solution is dichloromethane; The azophenylformyl chloride compounds include one or more of 4-(4-aminosulfonylphenylazo)benzoyl chloride, 4-phenylazobenzoyl chloride, and 4-[(4-methoxyphenyl)azo]benzoyl chloride.

[0010] Preferably, the ratio of the ammonia polymer, triethylamine, organic solvent, azophenylformyl chloride compound, and propionyl chloride is 1 mol: 0.1~5.9 mol: 20~40 mL: 0.005~0.45 mol: 0.02~5.25 mol; The concentration of the acyl chloride solution is 0.005~0.45 mol / mL.

[0011] Preferably, the mixing temperature in step 1) is 0°C and the time is 30 min; The amidation reaction is carried out at a temperature of 20-25°C for 12-20 hours.

[0012] Preferably, the organic ligand comprises one or more of 2-methylimidazolium, 1,3,5-benzenetricarboxylic acid, 2-aminoterephthalic acid, and sodium terephthalate; The concentration of the organic ligand solution is 3-4 mol / L; The metal salt includes one or more of zinc nitrate, zirconium chloride, ferric chloride, and aluminum nitrate; The concentration of the metal salt solution is 0.3~0.35 mol / L.

[0013] Preferably, the functional enzyme includes one or more of carbonic anhydrase, lipase, glucose oxidase, catalase, laccase, and horseradish catalase.

[0014] Preferably, the mixing ratio of the photoresponsive polymer with the functional enzyme, organic ligand, and metal salt is 15~30mg: 0.2~20mg: 201.97~1418.8mg: 73.2mg.

[0015] Preferably, the mixing reaction in step 2) is carried out at room temperature (25°C) for 5 to 8 hours.

[0016] The present invention also provides a MOF enzyme catalytic material with photomodulation properties prepared by the above preparation method.

[0017] The present invention also provides the application of the MOF enzyme catalytic material with photomodulation properties, which can be used to catalyze the hydrolysis of p-nitrophenylacetic acid ester, the redox reaction of hydrogen peroxide, or the oxidation of β-D-glucose.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a photoresponsive enzyme catalytic composite material and its preparation method, integrating high stability, intelligent controllability, easy recyclability, and strong versatility. This material utilizes the synergistic effect of the MOF rigid framework and flexible polymer to construct a multi-layered regulatory system that combines rigidity and flexibility, thereby achieving activity regulation of enzymes in confined environments. It has broad application prospects in fields such as intelligent biosensing, precision medicine, environmental remediation, and green synthesis. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0020] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] This invention proposes a method for preparing MOF enzyme catalytic materials with photomodulation properties, which includes the following steps: 1) An amidation reaction was carried out by mixing an ammonia polymer, triethylamine, an organic solvent, an acyl chloride solution, and propionyl chloride to obtain a photoresponsive polymer; 2) A MOF enzyme catalytic material with photo-regulated properties is obtained by mixing and reacting photoresponsive polymers with solutions of functional enzymes, water, organic ligands, and metal salts. The solute in the acyl chloride solution is an azophenylformyl chloride compound.

[0025] In this invention, the ammonia polymer includes one or more of branched polyethyleneimine (PEI), polyallylamine (PAAm), polyacrylamide (PAM), and hyperbranched polyetheramine (HPEA).

[0026] In this invention, the solvent in the acyl chloride solution is dichloromethane.

[0027] In this invention, the preferred method for mixing the ammonia polymer, triethylamine, organic solvent, acyl chloride solution, and propionyl chloride is to mix the ammonia polymer, triethylamine, and organic solvent, and then simultaneously add the acyl chloride solution and propionyl chloride under ice bath conditions, wherein the organic solvent may be dichloromethane.

[0028] In this invention, the azophenylformyl chloride compounds include one or more of 4-(4-aminosulfonylphenylazo)benzoyl chloride, 4-phenylazobenzoyl chloride, and 4-[(4-methoxyphenyl)azo]benzoyl chloride.

[0029] In this invention, the ratio of the ammonia polymer, triethylamine, organic solvent, azophenylformyl chloride compound, and propionyl chloride is 1 mol: 0.1~5.9 mol: 20~40 mL: 0.005~0.45 mol: 0.02~5.25 mol, preferably 1 mol: 0.5~5 mol: 22~38 mL: 0.01~0.4 mol: 0.1~5 mol, more preferably 1 mol: 1~4 mol: 25~35 mL: 0.1~0.3 mol: 1~4 mol, and even more preferably 1 mol: 2~3 mol: 28~30 mL: 0.15~0.25 mol: 2~3 mol.

[0030] In this invention, the concentration of the acyl chloride solution is 0.005~0.45 mol / mL, preferably 0.01~0.4 mol / mL, more preferably 0.1~0.3 mol / mL, and even more preferably 0.15~0.25 mol / mL.

[0031] In this invention, the mixing temperature in step 1) is 0°C and the time is 30 min.

[0032] In this invention, the amidation reaction is carried out at a temperature of 20-25°C for 12-20 hours.

[0033] In this invention, after the amidation reaction is completed, a step of separating and purifying the photoresponsive polymer is further included, and the specific operation is as follows: The volatile solvent was removed by rotary evaporation, and then the product was purified by fractionation using a dialysis membrane with a molecular weight cutoff of 6000 Da (two methanol replacements and five deionized water dialysiss, 12 hours each). The dialysate was collected and stirred in an ice-water bath for 1 hour. The water-soluble components were separated by centrifugation, and the supernatant was freeze-dried to obtain the photoresponsive polymer.

[0034] In this invention, the organic ligand includes one or more of 2-methylimidazole, 1,3,5-benzenetricarboxylic acid, 2-aminoterephthalic acid, and sodium terephthalate.

[0035] In this invention, the concentration of the organic ligand solution is 3~4 mol / L, preferably 3.2~3.8 mol / L, more preferably 3.4~3.6 mol / L, and even more preferably 3.5 mol / L.

[0036] In this invention, the metal salt includes one or more of zinc nitrate, zirconium chloride, ferric chloride, and aluminum nitrate.

[0037] In this invention, the concentration of the metal salt solution is 0.3~0.35 mol / L, preferably 0.31~0.34 mol / L, more preferably 0.32~0.34 mol / L, and even more preferably 0.33 mol / L.

[0038] In this invention, the functional enzyme includes one or more of carbonic anhydrase (CA), lipase (LPS), glucose oxidase (GOx), catalase (CAT), laccase (LAc), and horseradish catalase (HRP).

[0039] In this invention, the mixing ratio of the photoresponsive polymer with the functional enzyme, organic ligand, and metal salt is 15-30 mg: 0.2-20 mg: 201.97-1418.8 mg: 73.2 mg, preferably 18-28 mg: 0.5-15 mg: 403.93-1211.80 mg: 73.2 mg, more preferably 18-25 mg: 1-12 mg: 201.97-1009.83 mg: 73.2 mg, and even more preferably 20-23 mg: 5-10 mg: 605.90-807.86 mg: 73.2 mg.

[0040] In this invention, the temperature of the mixing reaction in step 2) is room temperature (25°C) and the time is 5~8h.

[0041] In this invention, after the mixing reaction in step 2) is completed, the step of separating and purifying the MOF enzyme catalytic material with photoregulatory properties is also included. The catalytic material is obtained by centrifuging, washing and drying the mixed reaction solution in sequence.

[0042] The present invention also provides a MOF enzyme catalytic material with photomodulation properties prepared by the above preparation method.

[0043] The present invention also provides the application of the MOF enzyme catalytic material with photomodulation properties, which can be used to catalyze the hydrolysis of p-nitrophenylacetic acid ester, the redox reaction of hydrogen peroxide, or the oxidation of β-D-glucose.

[0044] Before use, the MOF enzyme catalytic material with photomodulation properties was mixed with HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer solution and allowed to stand for 3 hours. Then, it was irradiated with 360 nm ultraviolet light and 410 nm visible light for 20 minutes, respectively.

[0045] Example 1 Mix 0.0279 mol of an ammonia polymer (branched polyethyleneimine), 0.0287 mmol of triethylamine, and 30 mL of organic solvent (dichloromethane). Then, simultaneously add 1 mL of 0.417 mol / L 4-phenylazobenzoyl chloride solution and 0.913 mL of 11.5 mol / L propionyl chloride under ice bath conditions at 0 °C. After the addition is complete, stir for 30 min under ice water bath conditions, then remove the ice water bath and react at room temperature (25 °C) for 16 h.

[0046] After the reaction was completed, the volatile solvent (dichloromethane) was evaporated by rotary evaporator. Then, the solution was purified by fractionation using a dialysis membrane with a molecular weight cutoff of 6000 Da (two methanol replacements and five deionized water dialysiss, 12 hours each). The dialysis fluid was collected and stirred in an ice-water bath for 1 hour. The water-soluble components were separated by centrifugation, and the supernatant was freeze-dried to obtain the photoresponsive polymer.

[0047] 20 mg of photoresponsive polymer was mixed with 5 mg of carbonic anhydrase and 1 mL of deionized water. Then, 4 mL of 3.456 mol / L organic ligand (2-methylimidazole) solution was added and mixed evenly. Then, 0.75 mL of 0.328 mol / L metal salt (zinc nitrate hexahydrate) solution was slowly added. After stirring and reacting for 6 h, the photoresponsive enzyme catalytic material was obtained by centrifugation, washing and drying.

[0048] Example 2 Mix 0.0279 mol of an ammonia polymer (branched polyethyleneimine), 0.0287 mol of triethylamine, and 30 mL of organic solvent (dichloromethane). Then, simultaneously add 1 mL of 0.417 mol / L 4-phenylazobenzoyl chloride solution and 0.913 mL of 11.5 mol / L propionyl chloride under ice bath conditions at 0 °C. After the addition is complete, stir for 30 min under ice water bath conditions, then remove the ice water bath and react at room temperature (25 °C) for 16 h.

[0049] After the reaction was completed, the volatile solvent (dichloromethane) was evaporated by rotary evaporator. Then, the solution was purified by fractionation using a dialysis membrane with a molecular weight cutoff of 6000 Da (two methanol replacements and five deionized water dialysiss, 12 hours each). The dialysis fluid was collected and stirred in an ice-water bath for 1 hour. The water-soluble components were separated by centrifugation, and the supernatant was freeze-dried to obtain the photoresponsive polymer.

[0050] 20 mg of photoresponsive polymer was mixed with 5 mg of carbonic anhydrase and 1 mL of deionized water. Then, 3 mL of 3.456 mol / L organic ligand (imidazolium-2-formaldehyde) solution was added and mixed evenly. Then, 4 mL of 0.328 mol / L metal salt (zinc nitrate hexahydrate) solution was slowly added. After stirring and reacting for 6 h, the photoresponsive enzyme catalytic material was obtained by centrifugation, washing and drying.

[0051] Example 3 Mix 0.0279 mol of an ammonia polymer (branched polyethyleneimine), 0.0287 mol of triethylamine, and 30 mL of organic solvent (dichloromethane). Then, simultaneously add 1 mL of 0.417 mol / L 4-(4-aminosulfonylphenylazo)benzoyl chloride solution and 0.913 mL of 11.5 mol / L propionyl chloride under ice bath conditions at 0 °C. After the addition is complete, stir for 30 min under ice water bath conditions, then remove the ice water bath and react at room temperature (25 °C) for 16 h.

[0052] After the reaction was completed, the volatile solvent (dichloromethane) was evaporated by rotary evaporator. Then, the solution was purified by fractionation using a dialysis membrane with a molecular weight cutoff of 6000 Da (two methanol replacements and five deionized water dialysiss, 12 hours each). The dialysis fluid was collected and stirred in an ice-water bath for 1 hour. The water-soluble components were separated by centrifugation, and the supernatant was freeze-dried to obtain the photoresponsive polymer.

[0053] 20 mg of photoresponsive polymer was mixed with 5 mg of carbonic anhydrase and 1 mL of deionized water. Then, 4 mL of 3.456 mol / L organic ligand (2-methylimidazole) solution was added and mixed evenly. Then, 5 mL of 0.328 mol / L metal salt (cobalt nitrate hexahydrate) solution was slowly added. After stirring and reacting for 6 h, the photoresponsive enzyme catalytic material was obtained by centrifugation, washing and drying.

[0054] Example 4 Mix 0.0279 mol of an ammonia polymer (branched polyethyleneimine), 0.0287 mol of triethylamine, and 30 mL of organic solvent (dichloromethane). Then, simultaneously add 1 mL of 0.417 mol / L 4-(4-aminosulfonylphenylazo)benzoyl chloride, 1 mL of 0.417 mol / L 4-phenylazobenzoyl chloride solution, and 0.913 mL of 11.5 mol / L propionyl chloride under ice bath conditions at 0°C. After the addition is complete, stir for 30 min under ice water bath conditions, then remove the ice water bath and react at room temperature (25°C) for 16 h.

[0055] After the reaction was completed, the volatile solvent (dichloromethane) was evaporated by rotary evaporator. Then, the solution was purified by fractionation using a dialysis membrane with a molecular weight cutoff of 6000 Da (two methanol replacements and five deionized water dialysiss, 12 hours each). The dialysis fluid was collected and stirred in an ice-water bath for 1 hour. The water-soluble components were separated by centrifugation, and the supernatant was freeze-dried to obtain the photoresponsive polymer.

[0056] 20 mg of photoresponsive polymer was mixed with 5 mg of carbonic anhydrase and 1 mL of deionized water. Then, 4 mL of 3.456 mol / L organic ligand (2-methylimidazole) solution was added and mixed evenly. Then, 0.75 mL of 0.328 mol / L metal salt (zinc nitrate hexahydrate) solution was slowly added. After stirring and reacting for 6 h, the photoresponsive enzyme catalytic material was obtained by centrifugation, washing and drying.

[0057] Example 5 Mix 0.0279 mol of an ammonia polymer (branched polyethyleneimine), 0.0287 mol of triethylamine, and 30 mL of organic solvent (dichloromethane). Then, simultaneously add 1 mL of 0.417 mol / L 4-(4-aminosulfonylphenylazo)benzoyl chloride, 1 mL of 0.417 mol / L 4-phenylazobenzoyl chloride solution, and 0.913 mL of 11.5 mol / L propionyl chloride under ice bath conditions at 0°C. After the addition is complete, stir for 30 min under ice water bath conditions, then remove the ice water bath and react at room temperature (25°C) for 16 h.

[0058] After the reaction was completed, the volatile solvent (dichloromethane) was evaporated by rotary evaporator. Then, the solution was purified by fractionation using a dialysis membrane with a molecular weight cutoff of 6000 Da (two methanol replacements and five deionized water dialysiss, 12 hours each). The dialysis fluid was collected and stirred in an ice-water bath for 1 hour. The water-soluble components were separated by centrifugation, and the supernatant was freeze-dried to obtain the photoresponsive polymer.

[0059] 20 mg of photoresponsive polymer was mixed with 5 mg of carbonic anhydrase and 1 mL of deionized water. Then, 3 mL of 3.456 mol / L organic ligand (imidazolium-2-formaldehyde) solution was added and mixed evenly. Then, 4 mL of 0.328 mol / L metal salt (zinc nitrate hexahydrate) solution was slowly added. After stirring and reacting for 6 h, the photoresponsive enzyme catalytic material was obtained by centrifugation, washing and drying.

[0060] Example 6 Mix 0.0279 mol of an ammonia polymer (branched polyethyleneimine), 0.0287 mol of triethylamine, and 30 mL of organic solvent (dichloromethane). Then, simultaneously add 1 mL of 0.417 mol / L 4-phenylazobenzoyl chloride solution and 0.913 mL of 11.5 mol / L propionyl chloride under ice bath conditions at 0 °C. After the addition is complete, stir for 30 min under ice water bath conditions, then remove the ice water bath and react at room temperature (25 °C) for 16 h.

[0061] After the reaction was completed, the volatile solvent (dichloromethane) was evaporated by rotary evaporator. Then, the solution was purified by fractionation using a dialysis membrane with a molecular weight cutoff of 6000 Da (two methanol replacements and five deionized water dialysiss, 12 hours each). The dialysis fluid was collected and stirred in an ice-water bath for 1 hour. The water-soluble components were separated by centrifugation, and the supernatant was freeze-dried to obtain the photoresponsive polymer.

[0062] 20 mg of photoresponsive polymer was mixed with 5 mg of lipase and 1 mL of deionized water. Then, 4 mL of 3.456 mol / L organic ligand (2-methylimidazole) solution was added and mixed evenly. Then, 5 mL of 0.328 mol / L metal salt (cobalt nitrate hexahydrate) solution was slowly added. After stirring and reacting for 6 h, the photoresponsive enzyme catalytic material was obtained by centrifugation, washing and drying.

[0063] Example 7 Mix 0.0279 mol of an ammonia polymer (branched polyethyleneimine), 0.0287 mol of triethylamine, and 30 mL of organic solvent (dichloromethane). Then, simultaneously add 1 mL of 0.417 mol / L 4-(4-aminosulfonylphenylazo)benzoyl chloride solution and 0.913 mL of 11.5 mol / L propionyl chloride under ice bath conditions at 0 °C. After the addition is complete, stir for 30 min under ice water bath conditions, then remove the ice water bath and react at room temperature (25 °C) for 16 h.

[0064] After the reaction was completed, the volatile solvent (dichloromethane) was evaporated by rotary evaporator. Then, the solution was purified by fractionation using a dialysis membrane with a molecular weight cutoff of 6000 Da (two methanol replacements and five deionized water dialysiss, 12 hours each). The dialysis fluid was collected and stirred in an ice-water bath for 1 hour. The water-soluble components were separated by centrifugation, and the supernatant was freeze-dried to obtain the photoresponsive polymer.

[0065] 20 mg of photoresponsive polymer was mixed with 5 mg of lipase and 1 mL of deionized water. Then, 4 mL of 3.456 mol / L organic ligand (2-methylimidazole) solution was added and mixed evenly. Then, 0.75 mL of 0.328 mol / L metal salt (zinc nitrate hexahydrate) solution was slowly added. After stirring and reacting for 6 h, the reaction was centrifuged, washed, and dried to obtain the photoresponsive enzyme catalytic material.

[0066] Example 8 Mix 0.0279 mol of an ammonia polymer (branched polyethyleneimine), 0.0287 mol of triethylamine, and 30 mL of organic solvent (dichloromethane). Then, simultaneously add 1 mL of 0.417 mol / L 4-(4-aminosulfonylphenylazo)benzoyl chloride solution and 0.913 mL of 11.5 mol / L propionyl chloride under ice bath conditions at 0 °C. After the addition is complete, stir for 30 min under ice water bath conditions, then remove the ice water bath and react at room temperature (25 °C) for 16 h.

[0067] After the reaction was completed, the volatile solvent (dichloromethane) was evaporated by rotary evaporator. Then, the solution was purified by fractionation using a dialysis membrane with a molecular weight cutoff of 6000 Da (two methanol replacements and five deionized water dialysiss, 12 hours each). The dialysis fluid was collected and stirred in an ice-water bath for 1 hour. The water-soluble components were separated by centrifugation, and the supernatant was freeze-dried to obtain the photoresponsive polymer.

[0068] 20 mg of photoresponsive polymer was mixed with 5 mg of lipase and 1 mL of deionized water. Then, 3 mL of 3.456 mol / L organic ligand (imidazolium-2-formaldehyde) solution was added and mixed evenly. Then, 4 mL of 0.328 mol / L metal salt (zinc nitrate hexahydrate) solution was slowly added. After stirring and reacting for 6 h, the photoresponsive enzyme catalytic material was obtained by centrifugation, washing and drying.

[0069] Example 9 Mix 0.0279 mol of an ammonia polymer (branched polyethyleneimine), 0.0287 mol of triethylamine, and 30 mL of organic solvent (dichloromethane). Then, simultaneously add 1 mL of 0.417 mol / L 4-(4-aminosulfonylphenylazo)benzoyl chloride, 1 mL of 0.417 mol / L 4-phenylazobenzoyl chloride solution, and 0.913 mL of 11.5 mol / L propionyl chloride under ice bath conditions at 0°C. After the addition is complete, stir for 30 min under ice water bath conditions, then remove the ice water bath and react at room temperature (25°C) for 16 h.

[0070] After the reaction was completed, the volatile solvent (dichloromethane) was evaporated by rotary evaporator. Then, the solution was purified by fractionation using a dialysis membrane with a molecular weight cutoff of 6000 Da (two methanol replacements and five deionized water dialysiss, 12 hours each). The dialysis fluid was collected and stirred in an ice-water bath for 1 hour. The water-soluble components were separated by centrifugation, and the supernatant was freeze-dried to obtain the photoresponsive polymer.

[0071] 20 mg of photoresponsive polymer was mixed with 5 mg of lipase and 1 mL of deionized water. Then, 4 mL of 3.456 mol / L organic ligand (2-methylimidazole) solution was added and mixed evenly. Then, 5 mL of 0.328 mol / L metal salt (cobalt nitrate hexahydrate) solution was slowly added. After stirring and reacting for 6 h, the photoresponsive enzyme catalytic material was obtained by centrifugation, washing and drying.

[0072] Table 1. Composition of the photoresponsive enzyme catalytic materials prepared in Examples 1-9 Wherein, Azo is a photoresponsive azobenzene, and LRI is an azobenzene inhibitor group.

[0073] The catalytic effects of the photoresponsive enzyme catalytic materials prepared in Examples 1-9 were tested. The test methods and results are as follows: p-Nitrophenylacetic acid (NPA) was used as the substrate for enzyme activity detection, and the catalytic activity was evaluated by monitoring its hydrolysis reaction process. A small amount of the photoresponsive catalytic material prepared in Examples 1-5 was added to HEPES buffer and allowed to stand for 3 hours. Then, it was irradiated with ultraviolet light (360 nm) and visible light (410 nm) for 20 minutes, respectively. A 750 μmol / L p-nitrophenylacetic acid solution was added, and the enzyme catalysis was tested at 400 nm using a UV / Vis spectrophotometer. The results are shown in Table 2.

[0074] Table 2 Catalytic effects of the photoresponsive catalytic materials prepared in Examples 1-5 Note: Relative enzyme activity is defined as 100% of the free enzyme activity under no-light conditions; The rate of change in relative enzyme activity = relative enzyme activity under ultraviolet light - relative enzyme activity under visible light.

[0075] A small amount of the photoresponsive catalytic material prepared in Examples 6-9 was added to HEPES buffer and allowed to stand for 3 hours. Then, it was irradiated with ultraviolet light (360 nm) and visible light (410 nm) for 20 minutes each. In the dark, 100 mmol / L sodium dihydrogen phosphate and 100 mmol / L ethyl p-nitrophenylacetate were added, and the mixture was incubated at 40°C for 5 minutes. Marmer solution (2.0 mL, chloroform:isoamyl alcohol, 24:1) was added. After centrifugation, the clear, pale yellow aqueous supernatant (p-nitrophenol) was collected, and the enzyme activity was measured at 405 nm using a UV / Vis spectrophotometer. The results are shown in Table 3. Table 3 Catalytic effects of the photoresponsive catalytic materials prepared in Examples 6-9 As can be seen from the data in Tables 2 and 3, the azobenzene-functionalized MOF immobilized enzyme materials prepared in this invention all possess reversible photoresponsive catalytic properties of UV activation and visible light inhibition. Furthermore, the simultaneous introduction of Azo photoresponsive groups and LRI inhibitor groups into the polymer produces a synergistic regulatory effect, significantly improving the photomodulation activity compared to single-functional polymers.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a MOF enzyme catalytic material with photomodulation properties, characterized in that, Includes the following steps: 1) An amidation reaction was carried out by mixing an ammonia polymer, triethylamine, an organic solvent, an acyl chloride solution, and propionyl chloride to obtain a photoresponsive polymer; 2) A MOF enzyme catalytic material with photo-regulated properties is obtained by mixing and reacting photoresponsive polymers with solutions of functional enzymes, water, organic ligands, and metal salts. The solute in the acyl chloride solution is an azophenylformyl chloride compound.

2. The method for preparing a MOF enzyme catalytic material with photomodulation properties according to claim 1, characterized in that, The ammonia polymers include one or more of branched polyethyleneimine, polyallylamine, polyacrylamide, and hyperbranched polyetheramine; The solvent in the acyl chloride solution is dichloromethane; The azophenylformyl chloride compounds include one or more of 4-(4-aminosulfonylphenylazo)benzoyl chloride, 4-phenylazobenzoyl chloride, and 4-[(4-methoxyphenyl)azo]benzoyl chloride.

3. The method for preparing a MOF enzyme catalytic material with photomodulation properties according to claim 1, characterized in that, The ratio of the ammonia polymer, triethylamine, organic solvent, azophenylformyl chloride compound, and propionyl chloride is 1 mol: 0.1~5.9 mol: 20~40 mL: 0.005~0.45 mol: 0.02~5.25 mol; The concentration of the acyl chloride solution is 0.005~0.45 mol / mL.

4. The method for preparing a MOF enzyme catalytic material with photomodulation properties according to claim 1, characterized in that, The mixing temperature in step 1) is 0℃, and the time is 30 minutes; The amidation reaction is carried out at a temperature of 20-25°C for 12-20 hours.

5. The method for preparing a MOF enzyme catalytic material with photomodulation properties according to claim 1, characterized in that, The organic ligand includes one or more of 2-methylimidazolium, 1,3,5-benzenetricarboxylic acid, 2-aminoterephthalic acid, and sodium terephthalate; The concentration of the organic ligand solution is 3-4 mol / L; The metal salt includes one or more of zinc nitrate, zirconium chloride, ferric chloride, and aluminum nitrate; The concentration of the metal salt solution is 0.3~0.35 mol / L.

6. The method for preparing a MOF enzyme catalytic material with photomodulation properties according to claim 1, characterized in that, The functional enzymes include one or more of carbonic anhydrase, lipase, glucose oxidase, catalase, laccase, and horseradish catalase.

7. The method for preparing a MOF enzyme catalytic material with photomodulation properties according to claim 1, characterized in that, The mixing ratio of the photoresponsive polymer with the functional enzyme, organic ligand, and metal salt is 15~30mg: 0.2~20mg: 201.97~1418.8mg: 73.2mg.

8. The method for preparing a MOF enzyme catalytic material with photomodulation properties according to claim 1, characterized in that, The mixing reaction in step 2) is carried out at room temperature (25°C) for 5-8 hours.

9. The MOF enzyme catalytic material with photomodulation properties prepared by the method of any one of claims 1 to 8.

10. The application of the MOF enzyme catalytic material with photomodulation properties as described in claim 9, characterized in that, It can be used to catalyze the hydrolysis of p-nitrophenylacetic acid ester, the redox reaction of hydrogen peroxide, or the oxidation of β-D-glucose.

Images