A protein-inorganic organic nanocomposite with high thermal stability and a preparation method thereof

Abstract

This invention discloses a protein-inorganic-organic nanocomposite with high thermal stability and its preparation method. The nanocomposite material is prepared by coordinating and self-assembling inorganic and organic components in a solvent. Simultaneously, enzymes and other biomacromolecules are added during the reaction to load the nanocomposite, resulting in the protein-inorganic-organic nanocomposite. The addition of a protein activity protectant during preparation gives the composite extremely high thermal stability; the resulting product maintains its properties under extreme high-temperature conditions above 180°C while effectively preserving the enzyme's biological activity.

Description

Technical Field

[0001] This invention relates to the fields of hybrid material preparation, immobilized enzymes and material processing technology, and more specifically, to a protein-inorganic-organic nanocomposite with high thermal stability and its preparation method. Background Technology

[0002] Enzymes are green biocatalysts. The vast majority of enzymes are proteins with specific spatial structures produced by living cells. Their catalytic reactions are characterized by mildness, high efficiency, and high selectivity. However, once removed from the cellular environment and placed in the context of actual industrial production, enzyme activity is often significantly affected. High temperatures, harsh pH conditions, and large amounts of organic solvents are common reaction conditions in chemical production, but they can destroy the spatial structure of enzymes, causing them to lose their catalytic activity and severely limiting their industrial application. Therefore, enzyme immobilization technology has attracted widespread attention. In recent years, many methods have been proposed for enzyme immobilization to improve their stability, such as chemical cross-linking, carrier adsorption, covalent bonding, and confined embedding. Furthermore, with the rapid development of materials synthesis technology, various high-performance and structurally diverse materials have been used as carriers for enzyme immobilization, including a series of inorganic and organic materials such as mesoporous silica, metal-organic frameworks, molecular sieves, gels, and block polymers. It has been found that immobilizing enzymes with these materials can effectively improve the thermal stability of enzymes in aqueous solutions, improve their tolerance to organic solvents, and increase their reusability.

[0003] Inorganic-organic nanohybrids can fully utilize the structural rigidity of inorganic components and the structural flexibility of organic components, making them excellent carriers for enzyme immobilization. Currently, many inorganic-organic nanohybrids are used for enzyme immobilization, such as metal-organic frameworks, and these materials have also been shown to effectively improve enzyme stability.

[0004] However, existing technologies rarely focus on the thermal stability of enzyme molecules outside of solvent environments at extremely high temperatures, such as above 100°C, an environment that is relevant to many industrial reactions, processing, and production processes. It is generally believed that temperatures above 100°C disrupt the conformation of enzyme molecules, and enzymes also lose essential water at this temperature, resulting in a near-complete loss of catalytic activity. In this patent, we propose an enzyme activity protection mechanism combining framework encapsulation, electrostatic modification, and chemical cross-linking, and based on this, we designed and synthesized a protein-inorganic-organic nanohybrid material for immobilizing enzyme molecules. By rationally designing the pore structure of the protein-inorganic-organic nanohybrid material, we maximize the amount of air within the framework, thereby reducing the overall thermal conductivity of the material. By adding a protein activity protectant, the interaction between the enzyme molecules and the carrier material is enhanced, thus effectively protecting the three-dimensional conformation of the enzyme in high-temperature environments. This invention exhibits good mechanical stability and biocompatibility, and can effectively protect enzyme activity at harsh temperatures above 180°C, which is of great significance for further expanding the application scenarios of enzymes. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a protein-inorganic-organic nanocomposite with high thermal stability, its preparation method, and its application.

[0006] In one aspect, the present invention provides a protein-inorganic-organic nanocomposite.

[0007] Furthermore, this protein-inorganic-organic nanocomposite is a porous material assembled from inorganic and organic components through coordination, and proteins and active protective agents are added during the preparation process.

[0008] Furthermore, the composite has a macroscopic form of a powdery solid and a microstructure of microspheres or polyhedral nanoparticles with a particle size distribution of 10-5000 nm. Preferably, its particle size is 10-500 nm.

[0009] Furthermore, the protein-inorganic-organic nanocomposite exhibits a pore size distribution of 1-100 nm and a specific surface area of ​​50-1300 m². 2 / g, pore volume 0.5-5.0cm 3 / g. Preferably, the protein-inorganic-organic nanocomposite has a pore size distribution of 1-20 nm, more preferably 1-5 nm, and a specific surface area of ​​800-1200 m². 2 / g, pore volume 0.5-1cm 3 / g.

[0010] Furthermore, the protein-inorganic-organic nanocomposite contains one or more inorganic metal elements, wherein the metal element content is 1-50 wt%, preferably 1 wt%, 2 wt%, 3 wt%, 5 wt%, 8 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, or 50 wt%, and the inorganic metal element is one or more of Fe, Co, Cr, Ca, Ni, Zn, Mn, Cu, and Ag.

[0011] Furthermore, the protein loading in this protein-inorganic-organic nanocomposite is 0.1-80 wt%, and it can effectively retain its biological activity at high temperatures. After heating at temperatures above 180°C for 0.5-20 min, the enzyme activity is retained at 10%-100%. Preferably, the protein loading in the protein-inorganic-organic nanocomposite is 0.1 wt%, 1 wt%, 2 wt%, 3 wt%, 5 wt%, 8 wt%, 10 wt%, 12 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt%, or 80 wt%, more preferably 1-30 wt%, and even more preferably 5-15 wt%.

[0012] In another aspect, the present invention provides a method for preparing a protein-inorganic-organic nanocomposite, comprising the following steps:

[0013] The protein-inorganic-organic nanocomposite was obtained by coordinating and self-assembling inorganic components, organic components, protein, and active protective agent in a solvent.

[0014] Preferably, the method includes adding inorganic components, organic components, proteins, and active protective agents to a solvent to obtain solutions containing inorganic components, organic components, proteins, and active protective agents respectively, then mixing them and performing a coordination self-assembly reaction to obtain the protein-inorganic-organic nanocomposite.

[0015] In this invention, the inorganic component is selected from metal salts, specifically from one or more salts of cobalt, zinc, iron, calcium, chromium, copper, manganese, nickel, and silver; the anion of the salt may be one or more of halide ions, sulfate ions, bisulfate ions, nitrate ions, and acetate ions, especially nitrate ions. Preferably, the salt is a water-soluble metal salt, such as one or more of cobalt nitrate, cobalt chloride, zinc nitrate, zinc acetate, ferric chloride, calcium chloride, chromium chloride, copper nitrate, manganese chloride, and silver nitrate, including their anhydrous and hydrated forms, but not limited thereto.

[0016] Preferably, the inorganic component is a soluble metal salt. Further, the soluble metal salt is one or more of cobalt nitrate hexahydrate, cobalt chloride hexahydrate, zinc nitrate hexahydrate, zinc acetate dihydrate, anhydrous ferric chloride, anhydrous calcium chloride, chromium chloride hexahydrate, copper nitrate trihydrate, and manganese chloride tetrahydrate, but is not limited thereto.

[0017] In this invention, the organic component is a polyfunctional compound, such as selected from one or more of 2,2'-bipyridine, 4,4'-bipyridine, 2-methylimidazolium, 2-imidazolium carbaldehyde, 4-methylimidazolium, 1-methylimidazolium, benzimidazole, 3-methyl-1,2,4-triazole, 1,4-terephthalic acid, 1,3,5-benzenetricarboxylic acid, 2,6-naphthalenedic acid, 4,4'-biphenyldicarboxylic acid, and butenedioic acid, but not limited thereto.

[0018] In this invention, the molar ratio of metal ions to organic components in the inorganic component is 1:0.01-100, preferably 1:0.1, 1:1, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:50, 1:100, and more preferably 1:5-50.

[0019] In this invention, the protein can be any one or more of the following enzymes: lipase, esterase, protease, keratinase, PETase, MHETase, cellulase, amylase, glycosidase, amide hydrolase, nitrile hydrolase, lysozyme, glucose oxidase, horseradish peroxidase, lactate oxidase, alcohol oxidase, cytochrome P450 oxidase, laccase, superoxide dismutase, catalase, formate dehydrogenase, glucose dehydrogenase, lactate dehydrogenase, D-amino acid dehydrogenase, ketone reductase, nitrile reductase, olefin reductase, etc., but is not limited thereto.

[0020] In this invention, the ratio of the protein to the metal ions in the inorganic components is 1-1000 mg:1 mmol, preferably 1 mg:1 mmol, 10 mg:1 mmol, 50 mg:1 mmol, 100 mg:1 mmol, 200 mg:1 mmol, 300 mg:1 mmol, 400 mg:1 mmol, 500 mg:1 mmol, 1000 mg:1 mmol, and more preferably 10-300 mg:1 mmol.

[0021] In this invention, the protein activity protectant is used to improve the stability of proteins at high temperatures. It consists of two parts: an electrostatic modifier and a chemical cross-linking agent. The synergistic effect of the two can significantly improve the protein protection activity.

[0022] The electrostatic modifier is any one or more of polyethylene glycol, polyvinylpyrrolidone, polyglutamic acid, polyaspartic acid, polylysine, branched polyethyleneimine, Pluronic F-127, and cysteine. Electrostatic modifiers can modify the surface of biomacromolecules such as enzymes, enhancing the repulsive effect between molecules by modifying their surface charge, thereby improving their stability.

[0023] The chemical crosslinking agent is one or more polyphenolic compounds, such as dopamine, tannic acid, and gallic acid. The chemical crosslinking agent can undergo oxidative self-polymerization under weakly alkaline conditions to form a coating on the material surface. Simultaneously, it can react with amino groups on the enzyme surface to form covalent crosslinks, thereby improving enzyme stability.

[0024] Further, the mass ratio of the electrostatic modifier to the protein is 1-100:100-1, preferably 1-100:100, specifically 1:100, 10:100, 20:100, 30:100, 40:100, 50:100, 60:100, 70:100, 80:100, 90:100, 100:100, and more preferably 20-50:100. Preferably, the mass ratio of the chemical cross-linking agent to the protein is 1-100:100-1, more preferably 1-100:100, specifically 1:100, 10:100, 20:100, 30:100, 40:100, 50:100, 60:100, 70:100, 80:100, 90:100, 100:100, and more preferably 20-50:100.

[0025] Furthermore, the solvent and the solvent in the solvent are selected from at least one of water, methanol, ethanol, and ammonia.

[0026] Furthermore, the coordination self-assembly reaction is carried out at a temperature of 0-40°C, preferably at room temperature; the reaction time is 0.1-48 h, preferably 0.5-2 h.

[0027] This invention provides a protein-inorganic-organic nanocomposite prepared by the above-described preparation method.

[0028] Compared with the prior art, the present invention has the following advantages and applications:

[0029] (1) The preparation method provided by the present invention is simple to operate and mild, and the prepared material has extremely high thermal stability and can effectively retain enzyme activity at temperatures above 180°C.

[0030] (2) Based on this, the present invention proposes an enzyme activity protection strategy of electrostatic modification + chemical crosslinking, which makes full use of the properties of various substances such as amino acids, polymers, and polyphenols, such as charge, functional groups, and reactivity, and uses them together as protein activity protectants. The synergistic effect of electrostatic modifiers and chemical crosslinking agents improves the protein protection ability.

[0031] (3) Due to the inorganic-organic framework structure and the addition of protein activity protectants, the resulting material can maintain stable physicochemical properties at extremely high temperatures and effectively protect protein activity at extremely high temperatures without solvents. Attached Figure Description

[0032] Figure 1 Here is a scanning electron microscope image of the protein-inorganic-organic nanocomposite from Example 6;

[0033] Figure 2 The XRD pattern of the protein-inorganic-organic nanocomposite in Example 6;

[0034] Figure 3 The nitrogen adsorption-desorption curve for Example 6;

[0035] Figure 4 This is a comparison of the stability of the free enzyme and the protein-inorganic-organic nanocomposite of Example 6 at 180°C. Detailed Implementation

[0036] The invention is described in more detail below to aid in understanding it.

[0037] It should be understood that the terms or words used in the specification and claims should not be construed as having the meaning defined in a dictionary, but rather as having a meaning consistent with their meaning in the context of the invention, based on the principle that the concept of a term may be appropriately defined by the inventor for the best explanation of the invention.

[0038] Example 1

[0039] This embodiment prepares an inorganic-organic nanocomposite without protein in an aqueous solution, including the following steps: (1) Prepare 1.0 mol / L of 1-methylimidazole aqueous solution, 0.1 mol / L of cobalt chloride aqueous solution, and 80 mg / mL of electrostatic modifier polyvinylpyrrolidone (PVP) aqueous solution, and sonicate at room temperature for 10 min until completely dissolved, wherein the molar ratio of cobalt ions to 1-methylimidazole is 1:10;

[0040] (2) Add 10 mL of cobalt chloride aqueous solution and 1 mL of PVP aqueous solution to 10 mL of 1-methylimidazole aqueous solution and stir for 30 min at room temperature using a magnetic stirrer with a speed of 500 rpm.

[0041] (3) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min, remove the supernatant and resuspend it with deionized water, and repeat the washing twice.

[0042] (4) The precipitate obtained in step (3) is pre-frozen at -20℃ for 12 hours and freeze-dried for 24 hours to obtain the inorganic-organic nanocomposite material, denoted as PVP-ZIF.

[0043] Example 2

[0044] This embodiment describes the preparation of a protein-free inorganic-organic nanocomposite in an aqueous solution, comprising the following steps:

[0045] (1) Prepare 1.0 mol / L ethanol solution of 1,3,5-benzenetricarboxylic acid, 0.1 mol / L aqueous solution of copper nitrate, 40 mg / mL aqueous solution of electrostatic modifier polyethylene glycol (PEG), and 40 mg / mL aqueous solution of chemical crosslinking agent tannic acid (TA). Sonicate at room temperature for 10 min until completely dissolved. The molar ratio of copper ions to 1,3,5-benzenetricarboxylic acid is 1:10.

[0046] (2) Add 10 mL of copper nitrate aqueous solution and 1 mL of PEG aqueous solution to 10 mL of ethanol solution of 1,3,5-benzenetricarboxylic acid, and stir for 30 min at room temperature using a magnetic stirrer with a speed of 500 rpm.

[0047] (3) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min, remove the supernatant and resuspend it with deionized water, and repeat the washing twice.

[0048] (4) The precipitate obtained in step (3) is pre-frozen at -20℃ for 12h and freeze-dried for 24h to obtain the inorganic-organic nanocomposite material, denoted as TA-PEG-HKU.

[0049] Example 3

[0050] This embodiment describes the preparation of a protein-free inorganic-organic nanocomposite in an aqueous solution, comprising the following steps:

[0051] (1) Prepare 1.0 mol / L of 4-methylimidazole aqueous solution, 0.1 mol / L of zinc acetate aqueous solution, 40 mg / mL of electrostatic modifier polyvinylpyrrolidone (PVP) aqueous solution, and 40 mg / mL of chemical crosslinking agent tannic acid (TA) aqueous solution. Sonicate at room temperature for 10 min until completely dissolved. The molar ratio of zinc ions to 4-methylimidazole is 1:10.

[0052] (2) Add 10 mL of zinc acetate aqueous solution, 1 mL of PVP aqueous solution and 1 mL of TA aqueous solution to 10 mL of 4-methylimidazole aqueous solution and stir for 30 min at room temperature using a magnetic stirrer with a speed of 500 rpm.

[0053] (3) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min, remove the supernatant and resuspend it with deionized water, and repeat the washing twice.

[0054] (4) The precipitate obtained in step (3) is pre-frozen at -20℃ for 12h and freeze-dried for 24h to obtain the inorganic-organic nanocomposite material, denoted as TA-PVP-ZIF.

[0055] Example 4

[0056] This embodiment describes the preparation of a protein-free inorganic-organic nanocomposite in an aqueous solution, comprising the following steps:

[0057] (1) Prepare 1.0 mol / L fumaric acid aqueous solution, 0.1 mol / L ferric chloride aqueous solution, 40 mg / mL electrostatic modifier polyvinylpyrrolidone (PVP) and 40 mg / mL chemical crosslinking agent dopamine (DA) aqueous solution respectively. Sonicate at room temperature for 10 min until completely dissolved. The molar ratio of ferric ions to fumaric acid is 1:10.

[0058] (2) Add 10 mL of ferric chloride aqueous solution, 1 mL of PVP aqueous solution and 1 mL of DA aqueous solution to 10 mL of fumaric acid aqueous solution and stir for 30 min at room temperature using a magnetic stirrer with a speed of 500 rpm.

[0059] (3) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min, remove the supernatant and resuspend it with deionized water, and repeat the washing twice.

[0060] (4) The precipitate obtained in step (3) is pre-frozen at -20℃ for 12h and freeze-dried for 24h to obtain the inorganic-organic nanocomposite material, denoted as DA-PVP-MIL.

[0061] Example 5

[0062] This embodiment describes the preparation of a protein-free inorganic-organic nanocomposite in an aqueous solution, comprising the following steps:

[0063] (1) Prepare 1.0 mol / L 2-methylimidazole aqueous solution, 0.1 mol / L cobalt nitrate aqueous solution, and 80 mg / mL tannic acid (TA) aqueous solution as a chemical crosslinking agent. Sonicate at room temperature for 10 min until completely dissolved. The molar ratio of cobalt ions to 2-methylimidazole is 1:10.

[0064] (2) Add 10 mL of cobalt nitrate aqueous solution and 1 mL of TA aqueous solution to 10 mL of 2-methylimidazole aqueous solution, and stir for 30 min at room temperature using a magnetic stirrer with a speed of 500 rpm.

[0065] (3) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min, remove the supernatant and resuspend it with deionized water, and repeat the washing twice.

[0066] (4) The precipitate obtained in step (3) is pre-frozen at -20℃ for 12h and freeze-dried for 24h to obtain the inorganic-organic nanocomposite material, denoted as TA-ZIF.

[0067] Example 6

[0068] This embodiment describes the preparation of protein-inorganic-organic nanocomposites in aqueous solution, including the following steps:

[0069] (1) Prepare 1.0 mol / L of 4-methylimidazole aqueous solution, 0.1 mol / L of zinc acetate aqueous solution, 40 mg / mL of electrostatic modifier polyvinylpyrrolidone (PVP) aqueous solution, 40 mg / mL of chemical crosslinking agent tannic acid (TA) aqueous solution and 100 mg / mL of protease aqueous solution respectively, and sonicate at room temperature for 10 min until completely dissolved. The molar ratio of zinc ions to 4-methylimidazole is 1:10.

[0070] (2) Add 10 mL of zinc acetate aqueous solution to 10 mL of 4-methylimidazole aqueous solution, mix well, and then add 1 mL of protease aqueous solution, 1 mL of PVP aqueous solution and 1 mL of TA aqueous solution. Stir at room temperature for 30 min using a magnetic stirrer with a speed of 500 rpm.

[0071] (3) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min, remove the supernatant and resuspend it with deionized water, and repeat the washing twice.

[0072] (4) The precipitate obtained in step (3) was pre-frozen at -20℃ for 12h and freeze-dried for 24h to obtain the inorganic-organic nanocomposite material loaded with protease, denoted as Pro@TA-PVP-ZIF;

[0073] (5) The protein loading rate in Pro@TA-PVP-ZIF is 8.8 wt%. Protein loading rate refers to the mass of protein contained in a unit mass of the complex, expressed in wt%, and can be calculated using the following formula:

[0074] Protein loading rate = (Amount of protein added - Amount of protein in supernatant and washing solution) / Mass of the resulting complex × 100%. Protein loading rates in subsequent examples will be calculated in this manner.

[0075] The microstructure of Pro@TA-PVP-ZIF was observed using scanning electron microscopy (see...). Figure 1 Its microstructure consists of uniformly distributed spherical nanoparticles with an average particle size of 45 nm. Its XRD pattern is shown below. Figure 2 As shown, typical ZIF diffraction peaks can be observed, confirming its spatially periodically ordered crystal structure. Its pore structure was analyzed using nitrogen adsorption spectroscopy, and the results are as follows. Figure 3 As shown, the calculated specific surface area of ​​Pro@TA-PVP-ZIF is 1008 m². 2 / g, pore volume is 0.63cm 3 / g, the pore size is mainly distributed in 1-3nm, and the average pore size is 2.6nm.

[0076] Example 7

[0077] This embodiment describes the preparation of protein-inorganic-organic nanocomposites in aqueous solution, including the following steps:

[0078] (1) Prepare 1.0 mol / L fumaric acid aqueous solution, 0.1 mol / L ferric chloride aqueous solution, 40 mg / mL electrostatic modifier polyvinylpyrrolidone (PVP) aqueous solution, 40 mg / mL chemical crosslinking agent dopamine (DA) aqueous solution and 100 mg / mL lipase aqueous solution respectively, and sonicate at room temperature for 10 min until completely dissolved. The molar ratio of ferric ions to fumaric acid is 1:10.

[0079] (2) First, mix 10 mL of ferric chloride aqueous solution, 1 mL of PVP aqueous solution, 1 mL of DA aqueous solution and 1 mL of lipase aqueous solution evenly, then add it to 10 mL of fumaric acid aqueous solution and stir for 30 min at room temperature using a magnetic stirrer with a speed of 500 rpm.

[0080] (3) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min, remove the supernatant and resuspend it with deionized water, and repeat the washing twice.

[0081] (4) The precipitate obtained in step (3) was pre-frozen at -20℃ for 12h and freeze-dried for 24h to obtain an inorganic-organic nanocomposite material loaded with lipase, denoted as Lip@DA-PVP-MIL.

[0082] (5) The protein loading rate in Lip@DA-PVP-MIL was 4.5 wt%.

[0083] Example 8

[0084] This embodiment describes the preparation of protein-inorganic-organic nanocomposites in aqueous solution, including the following steps:

[0085] (1) Prepare 1.0 mol / L of ethanol solution of 1,3,5-benzenetricarboxylic acid, 0.1 mol / L of aqueous solution of copper nitrate, 40 mg / mL of aqueous solution of electrostatic modifier polyethylene glycol (PEG), 40 mg / mL of aqueous solution of chemical crosslinking agent tannic acid (TA) and 100 mg / mL of aqueous solution of glucose oxidase (GOx), and sonicate at room temperature for 10 min until completely dissolved. The molar ratio of copper ions to 1,3,5-benzenetricarboxylic acid is 1:10.

[0086] (2) First, mix 10 mL of copper nitrate aqueous solution, 1 mL of PEG aqueous solution, 1 mL of TA aqueous solution and 1 mL of GOx aqueous solution evenly, then add it to 10 mL of ethanol solution of 1,3,5-benzenetricarboxylic acid, and stir for 30 min at room temperature using a magnetic stirrer with a speed of 500 rpm.

[0087] (3) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min, remove the supernatant and resuspend it with deionized water, and repeat the washing twice.

[0088] (4) The precipitate obtained in step (3) was pre-frozen at -20℃ for 12h and freeze-dried for 24h to obtain an inorganic-organic nanocomposite material loaded with glucose oxidase, denoted as GOx@TA-PEG-HKU.

[0089] (5) The protein loading rate in GOx@TA-PEG-HKU was 13.2 wt%.

[0090] Comparative Example 1

[0091] This comparative example prepares a protein-inorganic-organic nanocomposite in aqueous solution, but differs from Example 6 in that no protein activity protectant is added, and includes the following steps:

[0092] (1) Prepare 1.0 mol / L 4-methylimidazole aqueous solution, 0.1 mol / L zinc acetate aqueous solution and 100 mg / mL protease aqueous solution respectively, and sonicate at room temperature for 10 min until completely dissolved. The molar ratio of zinc ions to 4-methylimidazole is 1:10.

[0093] (2) Add 10 mL of zinc acetate aqueous solution to 10 mL of 4-methylimidazole aqueous solution, mix well, and then add 1 mL of protease aqueous solution. Stir at room temperature for 30 min using a magnetic stirrer with a speed of 500 rpm.

[0094] (3) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min, remove the supernatant and resuspend it with deionized water, and repeat the washing twice.

[0095] (4) The precipitate obtained in step (3) is pre-frozen at -20℃ for 12h and freeze-dried for 24h to obtain the inorganic-organic nanocomposite material loaded with protease, denoted as Pro@ZIF.

[0096] (5) The protein loading rate in Pro@ZIF was 3.2 wt%.

[0097] Comparative Example 2

[0098] This comparative example prepares a protein-inorganic-organic nanocomposite in an aqueous solution, but differs from Example 6 in that only an electrostatic modifier is added, without a chemical crosslinking agent, and includes the following steps:

[0099] (1) Prepare 1.0 mol / L 4-methylimidazole aqueous solution, 0.1 mol / L zinc acetate aqueous solution, 80 mg / mL electrostatic modifier polyvinylpyrrolidone (PVP) aqueous solution and 100 mg / mL protease aqueous solution respectively, and sonicate at room temperature for 10 min until completely dissolved. The molar ratio of zinc ions to 4-methylimidazole is 1:10.

[0100] (2) Add 10 mL of zinc acetate aqueous solution to 10 mL of 4-methylimidazole aqueous solution, mix well, and then add 1 mL of protease aqueous solution and 1 mL of PVP aqueous solution. Stir at room temperature for 30 min using a magnetic stirrer with a speed of 500 rpm.

[0101] (3) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min, remove the supernatant and resuspend it with deionized water, and repeat the washing twice.

[0102] (4) The precipitate obtained in step (3) was pre-frozen at -20℃ for 12h and freeze-dried for 24h to obtain the inorganic-organic nanocomposite material loaded with protease, denoted as Pro@PVP-ZIF.

[0103] (5) The protein loading rate in Pro@PVP-ZIF was 4 wt%.

[0104] Comparative Example 3

[0105] This comparative example prepares a protein-inorganic-organic nanocomposite in an aqueous solution, but differs from Example 6 in that only a chemical crosslinking agent is added, without an electrostatic modifier, and includes the following steps:

[0106] (1) Prepare 1.0 mol / L 4-methylimidazole aqueous solution, 0.1 mol / L zinc acetate aqueous solution, 80 mg / mL tannic acid (TA) aqueous solution and 100 mg / mL protease aqueous solution respectively, and sonicate at room temperature for 10 min until completely dissolved. The molar ratio of zinc ions to 4-methylimidazole is 1:10.

[0107] (2) Add 10 mL of zinc acetate aqueous solution to 10 mL of 4-methylimidazole aqueous solution, mix well, and then add 1 mL of protease aqueous solution and 1 mL of TA aqueous solution. Stir at room temperature for 30 min using a magnetic stirrer with a speed of 500 rpm.

[0108] (3) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min, remove the supernatant and resuspend it with deionized water, and repeat the washing twice.

[0109] (4) The precipitate obtained in step (3) was pre-frozen at -20℃ for 12h and freeze-dried for 24h to obtain the inorganic-organic nanocomposite material loaded with protease, denoted as Pro@TA-ZIF.

[0110] (5) The protein loading rate in Pro@TA-ZIF was 5.4 wt%.

[0111] Comparative Example 4

[0112] This comparative example prepares a protein-inorganic-organic nanocomposite in an aqueous solution, but differs from Example 7 in that no protein activity protectant is added, and includes the following steps:

[0113] (1) Prepare 1.0 mol / L fumaric acid aqueous solution, 0.1 mol / L ferric chloride aqueous solution, and 100 mg / mL lipase aqueous solution respectively, and sonicate at room temperature for 10 min until completely dissolved. The molar ratio of ferric ions to fumaric acid is 1:10.

[0114] (2) First, mix 10 mL of ferric chloride aqueous solution with 1 mL of lipase aqueous solution evenly, then add it to 10 mL of fumaric acid aqueous solution, and stir for 30 min at room temperature using a magnetic stirrer with a speed of 500 rpm.

[0115] (3) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min, remove the supernatant and resuspend it with deionized water, and repeat the washing twice.

[0116] (4) The precipitate obtained in step (3) is pre-frozen at -20℃ for 12h and freeze-dried for 24h to obtain an inorganic-organic nanocomposite material loaded with lipase, denoted as Lip@MIL.

[0117] (5) The protein loading rate in Lip@MIL was 4 wt%.

[0118] Comparative Example 5

[0119] This comparative example prepares a protein-inorganic-organic nanocomposite in an aqueous solution, but differs from Example 8 in that no protein activity protectant is added, and includes the following steps:

[0120] (1) Prepare 1.0 mol / L ethanol solution of 1,3,5-benzenetricarboxylic acid, 0.1 mol / L aqueous solution of copper nitrate and 100 mg / mL aqueous solution of glucose oxidase (GOx), and sonicate at room temperature for 10 min until completely dissolved. The molar ratio of copper ions to 1,3,5-benzenetricarboxylic acid is 1:10.

[0121] (2) First, mix 10 mL of copper nitrate aqueous solution with 1 mL of GOx aqueous solution evenly, then add it to 10 mL of ethanol solution of 1,3,5-benzenetricarboxylic acid, and stir for 30 min at room temperature using a magnetic stirrer with a speed of 500 rpm.

[0122] (3) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min, remove the supernatant and resuspend it with deionized water, and repeat the washing twice.

[0123] (4) The precipitate obtained in step (3) is pre-frozen at -20℃ for 12h and freeze-dried for 24h to obtain an inorganic-organic nanocomposite material loaded with glucose oxidase, denoted as GOx@HKU.

[0124] (5) The protein loading rate in GOx@HKU was 11.6 wt%.

[0125] The enzyme activity in protein-inorganic-organic nanocomposites loaded with hydrolytic enzymes such as proteases and lipases was determined using a UV-Vis spectrophotometer, including the following steps:

[0126] (1) Weigh 5 mg of the protein-inorganic-organic nanocomposite loaded with enzyme, add 1 mL of Tris-HCl buffer (80 mM, pH=7) containing 100 mM EDTA to hydrolyze the backbone, and stir with a magnetic stirrer at 500 rpm for 30 min.

[0127] (2) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min and retain the supernatant as the enzyme solution to be tested;

[0128] (3) Dissolve 2 μL of p-nitrophenol butyrate in 1 mL of acetone, and then add it dropwise to 20 mL of phosphate buffer (50 mM, pH = 7) under magnetic stirring, and continue stirring at 25°C for 5 min to obtain the assay substrate solution.

[0129] (4) In a typical assay system, 100 μL of the enzyme solution to be tested is added to 900 μL of the above-mentioned typical substrate solution, and the change in absorbance at 348 nm in the reaction system over time is detected. The specific enzyme solution concentration and detection time should be adjusted appropriately according to the different enzyme activities of the sample to ensure that the absorbance is within the linear range.

[0130] (5) Enzyme activity can be calculated using the following formula:

[0131] Enzyme activity = (ΔAbs / Δt) / concentration of the enzyme solution to be tested. The enzyme activity in a protein-inorganic-organic nanocomposite loaded with glucose oxidase was determined using a UV-Vis spectrophotometer, including the following steps:

[0132] (1) Weigh 5 mg of the protein-inorganic-organic nanocomposite loaded with enzyme, add 1 mL of Tris-HCl buffer (80 mM, pH=7) containing 100 mM EDTA to hydrolyze the backbone, and stir with a magnetic stirrer at 500 rpm for 30 min.

[0133] (2) Centrifuge the solution obtained in step (2) at 15000 rpm for 5 min and retain the supernatant as the enzyme solution to be tested;

[0134] (3) Add 20g of the supernatant enzyme solution to be tested, 80g of the solution with a concentration of 10%, and the horseradish peroxidase (HRP) solution to a substrate solution containing 100mM glucose and 0.5mM BTS at 900°C. Measure the change in absorbance at 415nm over time. The specific enzyme solution concentration and detection time should be adjusted appropriately according to the enzyme activity of the sample to ensure that the absorbance is within the linear range.

[0135] (4) Enzyme activity can be calculated using the following formula:

[0136] Enzyme activity = (ΔAbs / Δt) / concentration of the enzyme solution to be tested. The thermal stability of protein-inorganic-organic nanocomposites was determined using a small hot press, including the following steps:

[0137] (1) Weigh 10mg of protein-inorganic-organic nanocomposite, place it on a 30cm×30cm stainless steel sheet, and cover it with aluminum foil;

[0138] (2) Place the stainless steel sheet covered with aluminum foil between the upper and lower hot plates of the hot press, press the plates together, and heat at 180°C for different times (1 min, 2 min, 5 min, 10 min); (3) Release the pressure, remove the stainless steel sheet from between the upper and lower heating plates, tear off the covering aluminum foil, and recover the protein-inorganic-organic nanocomposite for enzyme activity determination.

[0139] Comparison of the appearance of samples (Example 6 and the corresponding free enzyme) before and after heat treatment. Figure 4 As shown, it can be seen that the properties of the free enzyme changed significantly after heating at 180°C, while the enzyme in Example 6 maintained its original properties after heating at 180°C.

[0140] The results of residual enzyme activity after heating at 180℃ are shown in Table 1 below. The residual enzyme activity represents the ratio of the activity of the protein-inorganic-organic nanocomposite before and after heating according to the aforementioned activity measurement method, i.e., activity after heating / activity before heating × 100%.

[0141] Table 1 Thermal stability of protein-inorganic-organic nanocomposites at 180℃

[0142]

[0143] Note: The activity assay for natural enzymes is to prepare a solution of the enzyme to be tested at a certain concentration in 1 mL of Tris-HCl buffer (80 mM, pH = 7) containing 100 mM EDTA. Other steps are the same as the activity assay for the complexes described above.

[0144] The preferred embodiments of the present invention have been described above, but are not intended to limit the invention. Those skilled in the art can make modifications and variations to the embodiments disclosed herein without departing from the scope and spirit of the invention.

Claims

1. A method for preparing a protein-inorganic-organic nanocomposite, comprising the following steps: Inorganic components, organic components, proteins, and active protective agents are added to solvents to obtain solutions containing inorganic components, organic components, proteins, and active protective agents, respectively. After mixing, coordination self-assembly reaction is carried out to obtain porous material protein-inorganic-organic nanocomposite. The inorganic component is selected from metal salts, specifically from one or more salts of cobalt, zinc, iron, calcium, chromium, copper, manganese, nickel, and silver; the anion of the salt is one or more of halide ions, sulfate ions, bisulfate ions, nitrate ions, and acetate ions. The organic component is a polyfunctional compound selected from one or more of the following: 2,2'-bipyridine, 4,4'-bipyridine, 2-methylimidazolium, 2-imidazolium carbaldehyde, 4-methylimidazolium, 1-methylimidazolium, benzimidazole, 3-methyl-1,2,4-triazole, 1,4-terephthalic acid, 1,3,5-benzenetricarboxylic acid, 2,6-naphthalenedic acid, 4,4'-biphenyldicarboxylic acid, and butenedioic acid; The protein is any one or more of the following: esterase, protease, PETase, MHETase, glycosidase, amide hydrolase, nitrile hydrolase, lysozyme, glucose oxidase, horseradish peroxidase, lactate oxidase, alcohol oxidase, cytochrome P450 oxidase, laccase, superoxide dismutase, catalase, formate dehydrogenase, glucose dehydrogenase, lactate dehydrogenase, D-amino acid dehydrogenase, ketone reductase, nitrile reductase, and olefin reductase. The active protective agent consists of two parts: an electrostatic modifier and a chemical crosslinking agent. The electrostatic modifier is any one or more of polyethylene glycol, polyvinylpyrrolidone, polyglutamic acid, polyaspartic acid, polylysine, branched polyethyleneimine, Pluronic F-127, and cysteine. The chemical crosslinking agent is any one or more of polyphenolic compounds, including dopamine, tannic acid, and gallic acid. The mass ratio of electrostatic modifier to protein is 1-100:1; the mass ratio of chemical cross-linking agent to protein is 1-100:

1. The protein loading in the complex ranges from 0.1 to 80 wt%.

2. The preparation method according to claim 1, characterized in that, The molar ratio of metal ions to organic components in the inorganic component is 1:0.01-100.

3. The preparation method according to claim 1, characterized in that, The ratio of the protein to the metal ions in the inorganic components is 1-1000 mg: 1 mmol.

4. A protein-inorganic-organic nanocomposite prepared by the method according to any one of claims 1-3, characterized in that, The macroscopic property of the composite is powder-like solid, and the microstructure is microspheres or polyhedral nanoparticles with particle size distribution of 10-5000 nm; the pore size distribution is 1-100 nm, the specific surface area is 50-1300 m 2 / g, and the pore volume is 0.5-5.0 cm 3 / g.

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