In-situ enzyme immobilization method for improving enzyme activity and application of in-situ enzyme immobilization method

Abstract

The invention belongs to the technical field of enzyme immobilization, and particularly relates to an in-situ enzyme immobilization method for improving enzyme activity and application of the in-situ enzyme immobilization method. The method comprises the following steps: mixing oxidoreductase with a metal ion solution to obtain an enzyme-metal ion mixed solution; mixing a nitrogen-containing organic molecule with a ligand to obtain a nitrogen-containing organic molecule-ligand mixed solution; and mixing the enzyme-metal ion mixed solution with the nitrogen-containing organic molecule-ligand mixed solution, and carrying out immobilization to obtain the in-situ immobilized enzyme. The method can significantly improve the catalytic activity of the immobilized enzyme, wherein the activity of the immobilized catalase is 2.3 times higher than that of the free enzyme. The prepared enzyme carrier shows excellent anti-interference capability, can tolerate external disturbance of heat, solvents, protease and the like, and has remarkable in-vitro active oxygen scavenging capability. The invention provides a simple and effective strategy which can be used for improving the enzyme catalysis efficiency and has a wide application prospect in a biological catalysis system.

Description

Technical Field

[0001] The invention belongs to the technical field of immobilized enzymes, and in particular relates to a method and application of in-situ immobilized enzymes for improving enzyme activity. Background Art

[0002] Enzymes are a class of proteins that can catalyze biochemical reactions. They have high specificity and catalytic activity and are widely used in multiple industrial fields. They play an important role in detergent cleaning ingredients, saccharification reactions in food processing, and diagnostic reagents in the pharmaceutical industry. However, enzymes are easily affected by environmental factors (such as temperature, pH value, solvents, etc.) during use, which can lead to reduced activity or ineffectiveness. Therefore, how to extend the service life of enzymes and improve their stability in industrial processes has become a key challenge in enzyme application technology.

[0003] Enzyme immobilization technology combines enzyme molecules with solid carriers to form immobilized enzymes, which can maintain their activity in multiple reactions and remain stable under some extreme reaction conditions. Enzyme immobilization not only helps to improve the stability of enzymes, but also improves their reusability and reduces production costs, thus becoming an effective strategy to overcome enzyme fragility.

[0004] Storing enzymes in protective matrices has been shown to be an effective strategy to overcome the fragility of enzymes. Due to their high porosity, various porous matrices (porous silica particles, calcium carbonate, calcium phosphate, hydrogels, etc.) have been used for enzyme protection and immobilization. In recent years, Metal-Organic Frameworks (MOFs) have gradually attracted widespread attention from academia and industry due to their unique characteristics such as high porosity, structural diversity, and adjustable pore structure. In particular, Zeolitic Imidazolate Frameworks (ZIFs), such as ZIF-8, ZIF-67, and ZIF-90, have become the preferred materials in enzyme immobilization research due to their good structural stability and adaptability to environmental fluctuations. ZIF materials can not only effectively support enzyme encapsulation, but also provide a suitable reaction environment to enhance the stability and catalytic performance of enzymes.

[0005] Various enzymes, including catalase (CAT), horseradish peroxidase (HRP), glucose oxidase (GOx), etc., have been successfully encapsulated in situ into ZIF structures and have shown good catalytic performance. However, although these enzymes are successfully fixed in the ZIF framework, existing immobilization methods still have certain limitations. For example, after enzyme fixation, activity tends to decline, and the ZIF framework may form some defects during the immobilization process, which significantly reduce the stability of the enzyme and limit its application effect. Therefore, how to improve the enzyme immobilization process and enhance the enzyme catalytic activity and stability has become a problem to be solved in the current enzyme immobilization field. Summary of the invention

[0006] The purpose of the present invention is to provide a method and application of in-situ enzyme immobilization for improving enzyme activity. The in-situ enzyme immobilization method can significantly improve the catalytic activity and stability of the immobilized enzyme.

[0007] The present invention provides a method for in-situ immobilization of an enzyme for improving enzyme activity, comprising the following steps:

[0008] mixing the oxidoreductase with the metal ion solution to obtain an enzyme-metal ion mixed solution;

[0009] mixing the nitrogen-containing organic molecule with the ligand to obtain a nitrogen-containing organic molecule-ligand mixed solution;

[0010] The enzyme-metal ion mixed solution is mixed with the nitrogen-containing organic molecule-ligand mixed solution for immobilization to obtain the in-situ immobilized enzyme.

[0011] As a preferred embodiment, the oxidoreductase comprises at least one of the following enzymes: catalase, glucose oxidase and horseradish peroxidase;

[0012] The mass volume ratio of the oxidoreductase to the metal ion solution is 0.8-1.2 mg:1 mL.

[0013] As a preferred embodiment, the metal ion solution includes the following metal ions: Zn 2+ 、Co 2+ or Ca 2+ ; The concentration of the metal ion solution is 0.04~0.5M.

[0014] As a preferred embodiment, the nitrogen-containing organic molecule includes amino acids or polypeptides;

[0015] The amino acid includes alanine, isoleucine, alanine, cysteine, aspartic acid, glutamic acid, lysine, arginine or histidine;

[0016] The polypeptide comprises trimeryl histidine, hexahistidine or nonahistidine.

[0017] As a preferred embodiment, the mass volume ratio of the nitrogen-containing organic molecule to the ligand is 1-5 mg:2 mL.

[0018] As a preferred embodiment, the ligand comprises an organic ligand or an anionic ligand;

[0019] The organic ligand includes 2-methylimidazole or 2-aldehyde imidazole; the anionic ligand includes sodium carbonate or disodium hydrogen phosphate.

[0020] As a preferred embodiment, the concentration of the ligand is 0.1-0.5M.

[0021] As a preferred embodiment, the immobilization temperature is 20-30°C; the immobilization pH is 6-9.

[0022] The invention also provides the in-situ immobilized enzyme prepared by the method.

[0023] The present invention also provides the in-situ immobilized enzyme prepared by the method or the use of the in-situ immobilized enzyme in preparing a reagent for removing active oxygen in cells.

[0024] Beneficial effects: The present invention provides a method for in-situ immobilized enzyme to enhance enzyme activity, the method comprising the following steps: mixing an oxidoreductase with a metal ion solution to obtain an enzyme-metal ion mixed solution; mixing a nitrogen-containing organic molecule with a ligand to obtain a nitrogen-containing organic molecule-ligand mixed solution; mixing the enzyme-metal ion mixed solution with the nitrogen-containing organic molecule-ligand mixed solution for immobilization to obtain the in-situ immobilized enzyme. The present invention achieves efficient enzyme immobilization by mixing an amino acid or a polypeptide with a ligand in an aqueous phase system, and then adding catalase, glucose oxidase, horseradish peroxidase and catalase-glucose oxidase double enzymes. The method significantly improves the catalytic activity of the immobilized enzyme by providing an excellent microenvironment for the enzyme, wherein the activity of the immobilized catalase is 2.3 times higher than that of the free enzyme. The prepared enzyme carrier (CAT&peptide@ZIF-8) exhibits excellent anti-interference ability, can tolerate external disturbances such as heat, solvents and proteases, maintains excellent stability in multiple cycles, and has significant in vitro active oxygen scavenging ability. The invention provides a simple and effective strategy for improving enzyme catalytic efficiency and has broad application prospects in biocatalytic systems.

[0025] The present invention utilizes the advantages of high porosity and mild synthesis conditions of ZIF (ZIF-8, ZIF-90 and ZIF-67), calcium carbonate (CaCO3) and hydroxyapatite (HAP), and adopts the strategy of amino acid / polypeptide doping to add a mixed solution of metal ions and enzyme premixes to a mixed solution containing amino acids / polypeptides and ligands to construct a biocatalyst with multiple enzyme immobilization. The added amino acids / polypeptides regulate the microenvironment of the enzyme, improve the binding ability of the enzyme to the substrate, and thus improve the catalytic activity of the immobilized enzyme. In addition, the protective layer of the porous matrix not only increases the tolerance of the enzyme to extreme environments such as high temperature, organic solvents and proteases, but also effectively improves the recycling performance of the enzyme. In addition, the CAT enzyme fixed by His6-doped ZIF-8 can efficiently capture reactive oxygen species in cells and alleviate the level of cellular inflammation.

[0026] The method of the present invention immobilizes the enzyme by doping amino acids / peptides, thereby creating a favorable microenvironment for the enzyme. The prepared CAT&4His6@ZIF-8, GOx&4His6@ZIF-8, HRP&4His6@ZIF-8 and (GOx+CAT)&4His6@ZIF-8 composite materials significantly improve the enzyme catalytic activity. The preparation method is simple, fast and under mild conditions.

[0027] The CAT&4His6@ZIF-8 and (GOx+CAT)&4His6@ZIF-8 composite materials prepared by the present invention not only increase the tolerance of natural enzymes to extreme environments, but also improve the recyclability of natural enzymes. The CAT&4His6@ZIF-8 prepared by the present invention achieves efficient removal of intracellular reactive oxygen species. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0029] Figure 1 Schematic diagram of the preparation method of enzyme & polypeptide @ porous material in Examples 1 to 7 of the present invention, wherein the enzyme in the figure is a single enzyme of catalase, a single enzyme of glucose oxidase, a single enzyme of horseradish peroxidase, or a double enzyme of catalase-glucose oxidase;

[0030] Figure 2 Comparison of enzyme activities in the CAT & amino acid @ ZIF-8 composite material in Experimental Example 1;

[0031] Figure 3 Comparison of enzyme activities in the CAT&peptide@ZIF-8 composite material in Experimental Example 2;

[0032] Figure 4Comparison of enzyme activities in the CAT&nHis6@ZIF-8 composite material in Experimental Example 3;

[0033] Figure 5 This is the scanning electron microscope morphology of the CAT&4His6@ZIF-8 composite material in Experimental Example 3;

[0034] Figure 6 The encapsulation efficiency of CAT enzyme by CAT@ZIF-8 in Experimental Example 3 and the CAT&4His6@ZIF-8 composite material obtained in Example 3;

[0035] Figure 7 The stability of the enzyme in the CAT&4His6@ZIF-8 composite material in Experimental Example 3;

[0036] Figure 8 The recyclability of the CAT&4His6@ZIF-8 composite material in Experimental Example 3;

[0037] Fig. 9 The ability of the CAT&4His6@ZIF-8 composite material in Experimental Example 3 to remove intracellular reactive oxygen species;

[0038] Fig.10 is the relative enzyme activity of CAT&4His6@porous material in Experimental Example 4;

[0039] Fig.11 is the relative enzyme activity of GOx&4His6@porous material in Experimental Example 5;

[0040] Fig.12 is the relative enzyme activity of HRP&4His6@porous material in Experimental Example 6;

[0041] Fig.13 is the relative enzyme activity of (GOx+CAT)&4His6@porous material in Experimental Example 7;

[0042] Fig.14 The stability of GOx&4His6@ZIF-8 and HRP&4His6@ZIF-8 in organic solvents in Experimental Example 6;

[0043] Fig.15 This is the stability of (GOx+CAT)&4His6@ZIF-8 in organic solvents in Experimental Example 7. DETAILED DESCRIPTION

[0044] The present invention provides a method for in-situ immobilization of an enzyme for improving enzyme activity, comprising the following steps:

[0045] mixing the oxidoreductase with the metal ion solution to obtain an enzyme-metal ion mixed solution;

[0046] mixing the nitrogen-containing organic molecule with the ligand to obtain a nitrogen-containing organic molecule-ligand mixed solution;

[0047] The enzyme-metal ion mixed solution is mixed with the nitrogen-containing organic molecule-ligand mixed solution for immobilization to obtain the in-situ immobilized enzyme.

[0048] As a specific embodiment, the oxidoreductase includes at least one of the following enzymes: catalase, glucose oxidase and horseradish peroxidase; in an embodiment of the present invention, the oxidoreductase is: 2 mg catalase (CAT), 2 mg glucose oxidase (GOx), 2 mg horseradish peroxidase (HRP) or a mixed enzyme of 2 mg glucose oxidase (GOx) + 1 mg catalase (CAT); as a specific embodiment, the in situ enzyme immobilization method of the present invention significantly improves the catalytic activity of the immobilized enzyme, wherein the activity of the immobilized catalase is 2.3 times higher than that of the free catalase.

[0049] As a specific embodiment, the metal ion solution includes the following metal ions: Zn 2+ 、Co 2+ or Ca 2 + ; The concentration of the metal ion solution is 0.04-0.5M; In an embodiment of the present invention, the metal ion solution is: 0.04M zinc acetate solution, 0.12M zinc nitrate solution, 0.05M cobalt nitrate solution, 0.1M calcium oxychloride solution or 0.5M calcium chloride solution.

[0050] The nitrogen-containing organic molecules of the present invention include amino acids or polypeptides; as a specific embodiment, the amino acids include phenylalanine (Phe), isoleucine (Ile), alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg) or histidine (His); as a specific embodiment, by testing the relative enzyme activity of immobilized enzymes doped with different amino acids, it was found that the relative enzyme activity of the immobilized enzymes doped with isoleucine Ile and glutamic acid Glu was the lowest, which were 9.7% and 9.2% respectively, and the relative enzyme activity of the immobilized enzyme doped with histidine was the highest, which was 107.7%.

[0051] As a specific embodiment, the polypeptide includes trimerized histidine (His3), hexamerized histidine (His6) or nonahistidine (His9). As a specific embodiment, by testing the relative enzyme activity of immobilized enzymes doped with different polypeptides, it was found that the immobilized enzyme doped with hexamerized histidine had the highest catalytic activity, with a relative enzyme activity of 231.1%. In the present invention, the mass of hexamerized histidine can be 1 mg, 2 mg, 4 mg and 5 mg. The test found that with the increase of the doping amount of hexamerized histidine, the relative enzyme activity of CAT&nHis6@ZIF-8 immobilized enzyme first increased and then decreased. The relative enzyme activity reached the maximum when the doping amount of hexamerized histidine was 4 mg, and its relative enzyme activity was 231.1%.

[0052] The ligand of the present invention includes an organic ligand or an anionic ligand; as a specific embodiment, the organic ligand includes 2-methylimidazole or 2-formyl imidazole; the anionic ligand includes sodium carbonate or disodium hydrogen phosphate. In an embodiment of the present invention, the ligand is 0.16M 2-methylimidazole, 0.5M 2-methylimidazole, 0.5M 2-formyl imidazole, 0.5M sodium carbonate or 0.1M disodium hydrogen phosphate. As a specific embodiment, the ligand of the present invention can form a metal ion-ligand with the metal ion solution, such as zinc ion-2-methylimidazole, zinc ion-2-formyl imidazole, cobalt ion-2-methylimidazole, calcium ion-sodium carbonate or calcium ion-disodium hydrogen phosphate, among which zinc ion-2-methylimidazole has the best fixation effect.

[0053] The immobilization temperature of the present invention is 20-30°C. As a specific embodiment, the immobilization temperature can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C; the immobilization pH is 6-9, such as 6, 7, 8 or 9. As a specific embodiment, the immobilization includes stirring and standing processes, and the stirring time can be 5min to 24h, such as 5min, 15min, 30min, 1h, 5h, 10h, 15h, 20h or 24h; the stirring speed can be 450rpm to 550rpm, such as 450rpm, 460rpm, 470rpm, 480rpm, 490rpm, 500rpm, 510rpm, 520rpm, 530rpm, 540rpm or 550rpm; the standing time can be 15min to 10h, such as 15min, 30min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h or 10h.

[0054] The invention also provides the in-situ immobilized enzyme prepared by the method.

[0055] The present invention also provides the in-situ immobilized enzyme prepared by the method or the use of the in-situ immobilized enzyme in preparing a reagent for removing active oxygen in cells.

[0056] The present invention utilizes the advantages of high porosity and mild synthesis conditions of ZIF (ZIF-8, ZIF-90 and ZIF-67), calcium carbonate (CaCO3) and hydroxyapatite (HAP), and adopts the strategy of amino acid / polypeptide doping to add a mixed solution of metal ions and enzyme premixes to a mixed solution containing amino acids / polypeptides and ligands to construct a biocatalyst with multiple enzyme immobilization. The added amino acids / polypeptides regulate the microenvironment of the enzyme, improve the binding ability of the enzyme to the substrate, and thus improve the catalytic activity of the immobilized enzyme. In addition, the protective layer of the porous matrix not only increases the tolerance of the enzyme to extreme environments such as high temperature, organic solvents and proteases, but also effectively improves the recycling performance of the enzyme. In addition, the CAT enzyme fixed by His6-doped ZIF-8 can efficiently capture reactive oxygen species in cells and alleviate the level of cellular inflammation.

[0057] To further illustrate the present invention, the method and application of an in situ immobilized enzyme for improving enzyme activity provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0058] Unless otherwise specified, the present invention has no special requirements for the preparation raw materials, and commercially available products known to those skilled in the art can be used.

[0059] Preparation of 0.04M zinc acetate solution and 0.16M 2-methylimidazole (2-MI) solution: weigh 878 mg of zinc acetate dihydrate and dilute to a 100mL volumetric flask at 25°C; weigh 1.314 g of 2-methylimidazole and dilute to a 100mL volumetric flask at 25°C.

[0060] Preparation of FOX working solution: ① First prepare 250mM sulfuric acid: add a portion of water to a beaker, then add 1.4mL concentrated sulfuric acid, and after the exotherm is complete, dilute to a 100mL volumetric flask. ② Prepare 100mM sorbitol: weigh 9.109g sorbitol and dissolve in water. ③ Prepare 100μM xylenol orange: weigh 35.8mg xylenol orange and dissolve in water. ④ Prepare 250μM ammonium ferrous sulfate: weigh 49.0mg ammonium ferrous sulfate and dissolve in 50mL of prepared ①. Add ② to ④, then add ③ to the mixed solution of ②④, dilute to a 500mL volumetric flask, and finally obtain FOX working solution.

[0061] Example 1

[0062] (1) Dissolve 2 mg of CAT (catalase) in 2 mL of 0.04 M zinc acetate solution and stir at 500 rpm for 1 min to obtain a mixed solution of CAT and zinc acetate.

[0063] (2) Dissolve 4 mg of amino acids (phenylalanine (Phe), isoleucine (Ile), alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg) or histidine (His)) in a mixed solution of 2 mL 0.16 M 2-methylimidazole and 30 μL triethylamine, and stir at 500 rpm for 1 min to obtain a mixed solution of 8 kinds of amino acids-2-methylimidazole-triethylamine.

[0064] (3) The mixed solution of CAT and zinc acetate in (1) was added to the mixed solution of 9 amino acids-2-methylimidazole-triethylamine in (2), respectively, stirred at room temperature and 500 rpm for 15 minutes, and allowed to stand for 8 hours to obtain 9 mixtures.

[0065] (4) The obtained 9 mixtures were centrifuged and washed three times with high-purity water, and the precipitates were collected to obtain 9 CAT&amino acid@ZIF-8 immobilized enzymes, namely CAT&Phe@ZIF-8 immobilized enzyme, CAT&Ile@ZIF-8 immobilized enzyme, CAT&Ala@ZIF-8 immobilized enzyme, CAT&Cys@ZIF-8 immobilized enzyme, CAT&Asp@ZIF-8 immobilized enzyme, CAT&Glu@ZIF-8 immobilized enzyme, CAT&Lys@ZIF-8 immobilized enzyme, CAT&Arg@ZIF-8 immobilized enzyme and CAT&His@ZIF-8 immobilized enzyme ( Figure 1 ).

[0066] Comparative Example 1

[0067] (1) Dissolve 2 mg of CAT in 2 mL of 0.16 M 2-methylimidazole solution and stir at 500 rpm for 1 min. Then add it to a mixed solution of 2 mL of 0.04 M zinc acetate and 30 μL of triethylamine, stir at room temperature at 500 rpm for 15 min, and let stand for 8 h.

[0068] (2) The obtained mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain CAT@ZIF-8 immobilized enzyme.

[0069] Experimental Example 1

[0070] Enzyme activity determination: FOX solution was used to detect the amount of H2O2 decomposition, and the relative enzyme activity of other immobilized materials was normalized and calculated using free CAT as a benchmark.

[0071] Take 500 μL of 150 μM H2O2 solution and mix it with 500 μL of CAT&amino acid@ZIF-8 immobilized enzyme solution (CAT&Phe@ZIF-8 immobilized enzyme, CAT&Ile@ZIF-8 immobilized enzyme, CAT&Ala@ZIF-8 immobilized enzyme, CAT&Cys@ZIF-8 immobilized enzyme, CAT&Asp@ZIF-8 immobilized enzyme, CAT&Glu@ZIF-8 immobilized enzyme, CAT&Lys@ZIF-8 immobilized enzyme, CAT&Arg@ZIF-8 immobilized enzyme or CAT&His@ZIF-8 immobilized enzyme) (enzyme content is 1 mg) prepared in Example 1; Take 500 μL of 150 μM H2O2 solution and mix it with 500 μL of CAT@ZIF-8 immobilized enzyme (enzyme content is 1 mg) prepared in Comparative Example 1; Take 500 μL of 150 μM H2O2 solution and 500 μL of After mixing for 120 seconds, take 50 μL of the reaction solution and mix it with 950 μL of FOX solution, keep it away from light for 30 minutes, and then measure the ultraviolet absorption value at 570 nm to obtain the relative activity value of the enzyme. Figure 2 The results show that the relative activity of the CAT@ZIF-8 immobilized enzyme prepared in Comparative Example 1 is 3.7% (the main reason is that the activity of the CAT enzyme is sensitive to the microenvironment, and ZIF-8 has a relatively hydrophobic microenvironment. The process of ZIF-8 encapsulating CAT will cause the conformation of CAT to change, making CAT inactive). Among the 9 CAT & amino acid @ZIF-8 immobilized enzymes prepared in Example 1, the relative enzyme activity of the immobilized enzyme doped with isoleucine Ile and glutamate Glu is the lowest, which are 9.7% and 9.2% respectively. The relative enzyme activity of the immobilized enzyme doped with histidine is the highest, which is 107.7%.

[0072] Example 2

[0073] (1) Dissolve 2 mg of CAT in 2 mL of 0.04 M zinc acetate solution and stir at 500 rpm for 1 min to obtain a mixed solution of CAT and zinc acetate.

[0074] (2) 4 mg of polypeptides of different chain lengths (trihistidine (His3), hexahistidine (His6) or nonahistidine (His9)) were dissolved in a mixed solution of 2 mL 0.16 M 2-methylimidazole and 30 μL triethylamine, respectively, and stirred at 500 rpm for 1 min to obtain a mixed solution of three amino acids-2-methylimidazole-triethylamine.

[0075] (3) The mixed solution of CAT and zinc acetate in (1) was added to the mixed solution of the three amino acids-2-methylimidazole-triethylamine in (2), respectively, and stirred at room temperature at 500 rpm for 15 min, and allowed to stand for 8 h. Figure 1), and 3 mixtures were obtained.

[0076] (4) The three mixtures were washed three times by centrifugation with high-purity water, and the precipitates were collected to obtain three CAT&polypeptide@ZIF-8 immobilized enzymes, namely CAT&His3@ZIF-8 immobilized enzyme, CAT&His6@ZIF-8 immobilized enzyme and CAT&His9@ZIF-8 immobilized enzyme.

[0077] Experimental Example 2

[0078] The enzyme activity of CAT&His3@ZIF-8 immobilized enzyme, CAT&His6@ZIF-8 immobilized enzyme and CAT&His9@ZIF-8 immobilized enzyme was determined according to the method of Experimental Example 1. The results are shown in Figure 3 , the hexahistidine-doped immobilized enzyme had the highest catalytic activity, with a relative enzyme activity of 231.1%.

[0079] Example 3

[0080] (1) Dissolve 2 mg of CAT in 2 mL of 0.04 M zinc acetate solution and stir at 500 rpm for 1 min to obtain a mixed solution of CAT and zinc acetate.

[0081] (2) Different masses of hexahistidine (1 mg, 2 mg, 4 mg or 5 mg) were dissolved in a mixed solution of 2 mL of 0.16 M 2-methylimidazole and 30 μL of triethylamine, and stirred at 500 rpm for 1 min to obtain a mixed solution of four amino acids-2-methylimidazole-triethylamine.

[0082] (3) The mixed solution of CAT and zinc acetate in (1) was added to the mixed solution of the four amino acids-2-methylimidazole-triethylamine in (2), respectively, and stirred at room temperature at 500 rpm for 15 min, and allowed to stand for 8 h. Figure 1 ), and 4 mixtures were obtained.

[0083] (4) The four mixtures were washed three times by centrifugation with high-purity water, and the precipitates were collected to obtain four CAT&nHis6@ZIF-8 immobilized enzymes, namely CAT&1His6@ZIF-8 immobilized enzyme, CAT&2His6@ZIF-8 immobilized enzyme, CAT&3His6@ZIF-8 immobilized enzyme and CAT&4His6@ZIF-8 immobilized enzyme.

[0084] Experimental Example 3

[0085] (1) According to the method of Experimental Example 1, the enzyme activity of four CAT&nHis6@ZIF-8 immobilized enzymes, namely CAT&1His6@ZIF-8 immobilized enzyme, CAT&2His6@ZIF-8 immobilized enzyme, CAT&3His6@ZIF-8 immobilized enzyme and CAT&4His6@ZIF-8 immobilized enzyme, was measured. The results are shown in Figure 4 With the increase of the hexahistidine doping amount, the relative enzyme activity of CAT&nHis6@ZIF-8 immobilized enzyme first increased and then decreased. When the hexahistidine doping amount was 4 mg, the relative enzyme activity reached the maximum, and its relative enzyme activity was 231.1%.

[0086] (2) Scanning electron microscopy observation of CAT&4His6@ZIF-8 showed the following results: Figure 5 As shown, CAT&4His6@ZIF-8 presents a dispersed rhombic dodecahedral structure.

[0087] (3) Encapsulation efficiency determination: The encapsulation contents of CAT@ZIF-8 and CAT&4His6@ZIF-8 were determined using a UV-visible-near infrared spectrophotometer and a 1260 Infinity II HPLC system in an acetonitrile / water (v:v=8:2) solution at a flow rate of 0.4 mL / min and a detection wavelength of 240 nm. Figure 6 ). As can be seen from the figure, the CAT enzyme encapsulation rates measured by the two methods are comparable, and the encapsulation rates of CAT@ZIF-8 and CAT&4His6@ZIF-8 are both around 90%.

[0088] (4) Enzyme stability test: Take 500 μL of CAT&4His6@ZIF-8, CAT@ZIF-8 and free CAT enzyme (enzyme content is 1 mg) respectively, and treat them at 50℃, 60℃ and 70℃ for 15 min, respectively; take 500 μL of CAT&4His6@ZIF-8, CAT@ZIF-8 and free CAT enzyme (enzyme content is 1 mg) respectively, and incubate them with equal volumes of MeOH, DMSO and CH2Cl2 solvents for 10 min, respectively; take 500 μL of CAT&4His6@ZIF-8, CAT@ZIF-8 and free CAT enzyme (enzyme content is 1 mg) respectively, and incubate them with 2 mg / mL proteinase K at 37℃ for 20 min. Then, the absorbance of CAT&4His6@ZIF-8, CAT@ZIF-8 and free CAT enzyme before and after treatment was determined using FOX solution, and the relative enzyme activity was calculated.

[0089] Depend on Figure 7 It can be seen that under the adverse conditions of high temperature, organic solvents and proteases, the activity of CAT&4His6@ZIF-8 can still be retained by more than 180%, while the free enzyme only retains less than 55% of its activity.

[0090] (5) CAT&4His6@ZIF-8 cycle performance test: In order to test the recyclability of the prepared CAT&4His6@ZIF-8, CAT&4His6@ZIF-8 was subjected to a freeze-thaw cycle test. 500μL of 2mg / mL CAT&4His6@ZIF-8 sample that had been frozen and thawed once was added to 500μL of H2O2. After the mixed solution reacted for 10 minutes, the absorbance of the system at 570nm was measured using FOX solution. The mixture was centrifuged at 5000rpm for 10 minutes to collect the precipitate for the next enzyme-catalyzed reaction. The process was repeated 10 times, and the relative enzyme activity was calculated using the change in catalytic activity before and after the FOX reaction system was recycled. Figure 8 It can be seen that CAT&4His6@ZIF-8 can still retain 180% of its activity after 10 cycles.

[0091] (6) CAT&4His6@ZIF-8 ability to remove intracellular reactive oxygen species: SH-sy5y cells (Saiye Biotechnology, H8-0101) were tested at 50000 / cm 2 The density of cells was seeded on a glass slide. After incubation in a cell culture incubator for 12 hours, blank control: no other reagents were added except for cells; 6-OHDA group: cells treated with 300 μM 6-hydroxydopamine hydrochloride (6-OHDA) (Aladdin reagent, H135753) were used as negative control group; 6-OHDA+CAT&4His6@ZIF-8 group: cells treated with 6-OHDA and CAT&4His6@ZIF-8 were used as experimental group; CAT&4His6@ZIF-8 group: cells treated with CAT&4His6@ZIF-8; After incubation for another 12 hours. The cells were washed twice with PBS, and then 10 μM 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (McLean reagent, D837204) probe was added and incubated in a 37°C incubator for 30 minutes. The cells were then washed twice with PBS, the cell nuclei were stained with DAPI, the cells were observed with a laser confocal microscope, and the relative fluorescence intensity was calculated to detect the material's ability to scavenge intracellular reactive oxygen species. Fig. 9 As shown, the fluorescence intensity of the 6-OHDA group was 100%, and the fluorescence intensity of the cells treated with CAT&4His6@ZIF-8 was 38.8%, which was equivalent to the fluorescence intensity of the blank control group (30.8%), significantly reducing the level of intracellular ROS.

[0092] Example 4

[0093] (1) Dissolve 0.5 M 2-formylimidazole and 4 mg hexahistidine in 2 mL of water to obtain a mixed aqueous solution of 2-formylimidazole-hexahistidine.

[0094] (2) Add 2 mg of CAT to 2 mL of 0.12 M zinc nitrate hexahydrate solution and stir at 500 rpm for 1 min to obtain a mixed solution of CAT and zinc nitrate.

[0095] (3) Add (1) to (2), stir for 30 min, and let stand at room temperature overnight. Wash the resulting mixture three times with high-purity water by centrifugation, collect the precipitate, and obtain CAT&4His6@ZIF-90 immobilized enzyme.

[0096] (4) Add 2 mg of CAT to 2 mL of 0.05 M cobalt nitrate solution and stir at 500 rpm for 1 min to obtain.

[0097] (5) Add (4) to 2 mL of a mixed solution containing 82.1 mg (0.5 M) 2-methylimidazole and 4 mg hexahistidine, stir for 30 min, and stand at room temperature overnight. The resulting mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain CAT&4His6@ZIF-67 immobilized enzyme.

[0098] (6) Add 2 mg of CAT to 2 mL of 0.5 M calcium chloride solution and stir at 1200 rpm for 1 min.

[0099] (7) Under stirring at 1200 rpm, 2 mL of a mixed solution containing 106 mg (0.5 M) sodium carbonate solution and 4 mg hexahistidine was added to (6) and stirred for 5 min, and then allowed to stand at room temperature for 15 min. The obtained mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain CAT&4His6@CaCO3 immobilized enzyme.

[0100] (8) Add 2 mg of CAT to 2 mL of 0.1 M calcium chloride solution and stir at 500 rpm for 1 min.

[0101] (9) Then, 2 mL of a mixed solution of pH 8.5 containing 28.4 mg of disodium hydrogen phosphate and 4 mg of hexahistidine was stirred and mixed with (8) at 37°C and 500 rpm for 24 h. The obtained mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain CAT&4His6@HAP immobilized enzyme.

[0102] Comparative Example 2

[0103] (1) Dissolve 0.5 M 2-formylimidazole in 2 mL of water to obtain an aqueous solution of 2-formylimidazole.

[0104] (2) Add 2 mg of CAT to 2 mL of 0.12 M zinc nitrate hexahydrate solution and stir at 500 rpm for 1 min.

[0105] (3) Add (1) to (2), stir for 30 min, and stand at room temperature overnight. Wash the resulting mixture three times with high-purity water by centrifugation, collect the precipitate, and obtain CAT@ZIF-90 immobilized enzyme.

[0106] (4) Add 2 mg of CAT to 2 mL of 0.05 M cobalt nitrate solution and stir at 500 rpm for 1 min.

[0107] (5) Add (4) to 2 mL of 0.5 M 2-methylimidazole solution, stir for 30 min, and stand at room temperature overnight. The resulting mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain CAT@ZIF-67 immobilized enzyme.

[0108] (6) Add 2 mg of CAT to 2 mL of 0.5 M calcium chloride solution and stir at 1200 rpm for 1 min.

[0109] (7) Add 2 mL of 0.5 M sodium carbonate solution to (6) and stir for 5 min at 1200 rpm, then let stand at room temperature for 15 min. Wash the resulting mixture three times with high-purity water by centrifugation, collect the precipitate, and obtain CAT@CaCO3 immobilized enzyme.

[0110] (8) Add 2 mg of CAT to 2 mL of 0.1 M calcium chloride solution and stir at 500 rpm for 1 min.

[0111] (9) Then, 2 mL of pH 8.5 0.1 M sodium dihydrogen phosphate solution was stirred and mixed with (8) at 37°C and 500 rpm for 24 min. The obtained mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain CAT@HAP immobilized enzyme.

[0112] Experimental Example 4

[0113] According to the method of Experimental Example 1, the enzyme activity of the CAT&4His6@ZIF-90 immobilized enzyme, CAT&4His6@ZIF-67 immobilized enzyme, CAT&4His6@CaCO3 immobilized enzyme, and CAT&4His6@HAP immobilized enzyme obtained in Example 4 and the CAT@ZIF-90 immobilized enzyme, CAT@ZIF-67 immobilized enzyme, CAT@CaCO3 immobilized enzyme, and CAT@HAP immobilized enzyme obtained in Comparative Example 2 was measured. The results are shown in Fig.10As can be seen from the figure, the activity of the immobilized enzyme directly mineralized by porous materials ZIF-8, ZIF-90, and ZIF-67 decreased significantly, by 3.7%, 71.3%, and 20.1%, respectively. After doping with hexahistidine, the enzyme activity increased significantly. The relative enzyme activity of the CAT&4His6@porous material immobilized enzyme was equivalent to that of the free enzyme, among which the CAT&4His6@ZIF-8 immobilized enzyme had the highest relative enzyme activity.

[0114] Example 5

[0115] (1) Dissolve 2 mg GOx (glucose oxidase) in 2 mL 0.04 M zinc acetate solution and stir at 500 rpm for 1 min.

[0116] (2) Dissolve 4 mg of hexahistidine in a mixed solution of 2 mL of 0.16 M 2-methylimidazole and 30 μL of triethylamine, and stir at 500 rpm for 1 min.

[0117] (3) The mixed solution of GOx and zinc acetate in (1) was added to the mixed solution of hexahistidine-2-methylimidazole-triethylamine (2), stirred at room temperature at 500 rpm for 15 min, and allowed to stand for 8 h.

[0118] (4) The obtained mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain GOx&4His6@ZIF8 immobilized enzyme.

[0119] (5) Dissolve 0.5 M 2-formylimidazole and 4 mg hexahistidine in 2 mL of water to obtain a mixed aqueous solution of 2-formylimidazole-hexahistidine.

[0120] (6) Add 2 mg of GOx into 2 mL of 0.12 M zinc nitrate hexahydrate solution and stir at 500 rpm for 1 min.

[0121] (7) Add (5) to (6), stir for 30 min, and let stand at room temperature overnight. The resulting mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain GOx&4His6@ZIF-90 immobilized enzyme.

[0122] (8) 2 mg of GOx was added to 2 mL of 0.05 M cobalt nitrate solution and stirred at 500 rpm for 1 min.

[0123] (9) Add (8) to 2 mL of a mixed solution containing 82.1 mg 2-methylimidazole and 4 mg hexahistidine, stir for 30 min, and stand at room temperature overnight. The resulting mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain GOx&4His6@ZIF-67 immobilized enzyme.

[0124] (10) 2 mg of GOx was added to 2 mL of 0.5 M calcium chloride solution and stirred at 1200 rpm for 1 min.

[0125] (11) Under stirring at 1200 rpm, 2 mL of a mixed solution containing 106 mg of sodium carbonate solution and 4 mg of hexahistidine was added to (10) and stirred for 5 min, and then allowed to stand at room temperature for 15 min. The obtained mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain GOx&4His6@CaCO3 immobilized enzyme.

[0126] (12) 2 mg of GOx was added to 2 mL of 0.1 M calcium chloride solution and stirred at 500 rpm for 1 min.

[0127] (13) Then, 2 mL of a mixed solution of pH 8.5 containing 28.4 mg of disodium hydrogen phosphate and 4 mg of hexahistidine was stirred and mixed with (12) at 37°C and 500 rpm for 24 h. The obtained mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain GOx&4His6@HAP immobilized enzyme.

[0128] Comparative Example 3

[0129] (1) Dissolve 2 mg GOx in 2 mL 0.04 M zinc acetate solution and stir at 500 rpm for 1 min.

[0130] (2) Add (1) to a mixed solution of 2 mL 0.16 M 2-methylimidazole and 30 μL triethylamine, stir at room temperature and 500 rpm for 15 min, and let stand for 8 h.

[0131] (3) The obtained mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain GOx@ZIF 8 immobilized enzyme.

[0132] (4) Dissolve 0.5 M 2-formylimidazole in 2 mL of water to obtain an aqueous solution of 2-formylimidazole.

[0133] (5) Add 2 mg of GOx into 2 mL of 0.12 M zinc nitrate hexahydrate solution and stir at 500 rpm for 1 min.

[0134] (6) Add (4) to (5), stir for 30 min, and let stand at room temperature overnight. The resulting mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain GOx@ZIF-90 immobilized enzyme.

[0135] (7) Add 2 mg of GOx into 2 mL of 0.05 M cobalt nitrate solution and stir at 500 rpm for 1 min.

[0136] (8) Add (7) to 2 mL of 0.5 M 2-methylimidazole solution, stir for 30 min, and stand at room temperature overnight. The resulting mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain GOx@ZIF-67 immobilized enzyme.

[0137] (9) Add 2 mg of GOx into 2 mL of 0.5 M calcium chloride solution and stir at 1200 rpm for 1 min.

[0138] (10) Add 2 mL of 0.5 M sodium carbonate solution to (9) and stir for 5 min at 1200 rpm, then let stand at room temperature for 15 min. Wash the resulting mixture three times with high-purity water by centrifugation, collect the precipitate, and obtain GOx@CaCO3 immobilized enzyme.

[0139] (11) 2 mg of GOx was added to 2 mL of 0.1 M calcium chloride solution and stirred at 500 rpm for 1 min.

[0140] (12) Then, 2 mL of pH 8.5 0.1 M sodium dihydrogen phosphate solution was stirred and mixed with (11) at 37°C and 500 rpm for 24 h. The obtained mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain GOx@HAP immobilized enzyme.

[0141] Experimental Example 5

[0142] Enzyme activity determination: 500 μL of 10 mg / mL glucose was added to 500 μL of 1 mg of the immobilized enzyme obtained in Example 5 (GOx&4His6@ZIF 8 immobilized enzyme, GOx&4His6@ZIF-90 immobilized enzyme, GOx&4His6@ZIF-67 immobilized enzyme, GOx&4His6@CaCO3 immobilized enzyme, GOx&4His6@HAP immobilized enzyme) and the immobilized enzyme obtained in Comparative Example 3 (GOx@ZIF 8 immobilized enzyme, GOx@ZIF-90 immobilized enzyme, GOx@ZIF-67 immobilized enzyme, GOx@CaCO3 immobilized enzyme, GOx@HAP immobilized enzyme) and 500 μL of 1 mg of free GOx sample to participate in the reaction. In a constant temperature water bath at 37°C, react for 30 minutes. At this time, glucose oxidase converts glucose into gluconic acid and generates hydrogen peroxide. After the reaction is completed, TMB solution is added. TMB will react with hydrogen peroxide to produce a blue compound. By measuring the absorbance at 450nm, the amount of hydrogen peroxide generated can be reflected, thereby obtaining the relative activity value of glucose oxidase. The results are as follows Fig.11As shown in the figure, compared with the directly mineralized GOx@ZIF-8 / ZIF-90 / ZIF-67, the corresponding relative enzyme activity was significantly improved after doping with hexahistidine, increasing to 198.7%, 126.8% and 57.6%, respectively. The relative enzyme activities of GOx&4His6@CaCO3 immobilized enzyme and GOx&4His6@HAP immobilized enzyme were comparable to those of GOx@CaCO3 immobilized enzyme and GOx@HAP.

[0143] Example 6

[0144] (1) Dissolve 0.5 M 2-formylimidazole and 4 mg hexahistidine in 2 mL of water to obtain a mixed aqueous solution of 2-formylimidazole-hexahistidine.

[0145] (2) Add 2 mg of HRP (horseradish peroxidase) to 2 mL of 0.12 M zinc nitrate hexahydrate solution and stir at 500 rpm for 1 min.

[0146] (3) Add (1) to (2), stir for 30 min, and let stand at room temperature overnight. Wash the resulting mixture three times with high-purity water by centrifugation, collect the precipitate, and obtain HRP&4His6@ZIF-90 immobilized enzyme.

[0147] (4) Add 2 mg of HRP to 2 mL of 0.05 M cobalt nitrate solution and stir at 500 rpm for 1 min.

[0148] (5) Add (4) to 2 mL of a mixed solution containing 82.1 mg of 2-methylimidazole and 4 mg of hexahistidine, stir for 30 min, and stand at room temperature overnight. The resulting mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain HRP&4His6@ZIF-67 immobilized enzyme.

[0149] (6) Add 2 mg of HRP to 2 mL of 0.5 M calcium chloride solution and stir at 1200 rpm for 1 min.

[0150] (7) Under stirring at 1200 rpm, 2 mL of a mixed solution containing 106 mg of sodium carbonate solution and 4 mg of hexahistidine was added to (6) and stirred for 5 min, and then allowed to stand at room temperature for 15 min. The obtained mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain HRP&4His6@CaCO3 immobilized enzyme.

[0151] (8) Add 2 mg of HRP to 2 mL of 0.1 M calcium chloride solution and stir at 500 rpm for 1 min.

[0152] (9) Then, 2 mL of a mixed solution of pH 8.5 containing 28.4 mg of disodium hydrogen phosphate and 4 mg of hexahistidine was stirred and mixed with (8) at 37°C and 500 rpm for 24 h. The obtained mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain the HRP&4His6@HAP immobilized enzyme.

[0153] (10) Dissolve 2 mg of HRP in 2 mL of 0.04 M zinc acetate solution and stir at 500 rpm for 1 min.

[0154] (11) Dissolve 4 mg of hexahistidine in a mixed solution of 2 mL of 0.16 M 2-methylimidazole and 30 μL of triethylamine and stir at 500 rpm for 1 min.

[0155] (12) Add (10) to (11), stir for 15 min, and stand at room temperature for 8 h. Wash the resulting mixture three times with high-purity water by centrifugation, and collect the precipitate to obtain HRP&4His6@ZIF-8 immobilized enzyme.

[0156] Comparative Example 4

[0157] (1) Dissolve 0.5 M 2-formylimidazole in 2 mL of water to obtain an aqueous solution of 2-formylimidazole.

[0158] (2) Add 2 mg of HRP to 2 mL of 0.12 M zinc nitrate hexahydrate solution and stir at 500 rpm for 1 min.

[0159] (3) Add (1) to (2), stir for 30 min, and let stand at room temperature overnight. Wash the obtained mixture by centrifugation with high-purity water for 3 times, collect the precipitate, and obtain HRP@ZIF-90 immobilized enzyme.

[0160] (4) Add 2 mg of HRP to 2 mL of 0.05 M cobalt nitrate solution and stir at 500 rpm for 1 min.

[0161] (5) Add (4) to 2 mL of 0.5 M 2-methylimidazole solution, stir for 30 min, and stand at room temperature overnight. Wash the resulting mixture three times with high-purity water by centrifugation, collect the precipitate, and obtain HRP@ZIF-67 immobilized enzyme.

[0162] (6) Add 2 mg of HRP to 2 mL of 0.5 M calcium chloride solution and stir at 1200 rpm for 1 min.

[0163] (7) Add 2 mL of 0.5 M sodium carbonate solution to (6) and stir for 5 min at 1200 rpm, then let stand at room temperature for 15 min. Wash the resulting mixture three times with high-purity water by centrifugation, collect the precipitate, and obtain HRP@CaCO3 immobilized enzyme.

[0164] (8) Add 2 mg of HRP to 2 mL of 0.1 M calcium chloride solution and stir at 500 rpm for 1 min.

[0165] (9) Then, 2 mL of pH 8.5 0.1 M sodium dihydrogen phosphate solution was stirred and mixed with (8) at 37°C and 500 rpm for 24 h. The obtained mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain HRP@HAP immobilized enzyme.

[0166] (10) Dissolve 2 mg of HRP in 2 mL of 0.04 M zinc acetate solution and stir at 500 rpm for 1 min.

[0167] (11) (10) was mixed with a mixed solution of 2 mL 0.16 M 2-methylimidazole and 30 μL triethylamine to obtain HRP@ZIF-8 immobilized enzyme.

[0168] Experimental Example 6

[0169] (1) Determination of enzyme activity: 500 μL of 150 μM H2O2 solution was mixed with 500 μL of 10 μM Amplex Red fluorescent red dye, and then the immobilized enzymes obtained in Example 6 (HRP & 4His6 @ ZIF-90 immobilized enzyme, HRP & 4His6 @ ZIF-67 immobilized enzyme, HRP & 4His6 @ CaCO3 immobilized enzyme, HRP & 4His6 @ HAP immobilized enzyme, HRP & 4His6 @ ZIF-8 immobilized enzyme) and the immobilized enzymes obtained in Comparative Example 4 (HRP @ ZIF-90 immobilized enzyme, HRP @ ZIF-67 immobilized enzyme, HRP @ CaCO3 immobilized enzyme, HRP @ HAP immobilized enzyme, HRP @ ZIF-8 immobilized enzyme) and an equal amount of free HRP (the mass of HRP enzyme is 1 mg) were added, and the fluorescence value at 585 nm was tested after 120 seconds of reaction to obtain the relative activity value of the enzyme. The results are as follows: Fig.12 As shown in the figure, compared with the directly mineralized HRP@ZIF-8 / ZIF-90 / ZIF-67, the corresponding relative enzyme activity was significantly improved after doping with hexahistidine, increasing to 199.5%, 72.1% and 122.9%, respectively. The relative enzyme activity of HRP&4His6@CaCO3 immobilized enzyme and HRP&4His6@HAP immobilized enzyme is comparable to that of HRP@CaCO3 immobilized enzyme and HRP@HAP.

[0170] (2) Enzyme stability test: 500 μL of GOx&4His6@ZIF-8 containing 1 mg enzyme, 1 mg enzyme of HRP&4His6@ZIF-8, 1 mg enzyme of free GOx enzyme, and 1 mg enzyme of free HRP enzyme were incubated with 500 μL of MeOH, DMSO, and CH2Cl2 solvents for 10 min, respectively. The absorbance before and after each enzyme treatment was measured using TMB solution, and the relative enzyme activity was calculated.

[0171] Depend on Fig.14 It can be seen that in an organic solvent environment, the activity of GOx&4His6@ZIF-8 can still be retained by more than 155%, while the free enzyme only retains less than 10% of its activity. In an organic solvent environment, the activity of HRP&4His6@ZIF-8 can still be retained by more than 130%, while the free enzyme only retains less than 15% of its activity.

[0172] Example 7

[0173] (1) Dissolve 1 mg CAT and 2 mg GOx in 2 mL 0.04 M zinc acetate solution and stir at 500 rpm for 1 min.

[0174] (2) Dissolve 4 mg of hexahistidine in a mixed solution of 2 mL of 0.16 M 2-methylimidazole and 30 μL of triethylamine, and stir at 500 rpm for 1 min.

[0175] (3) The mixed solution of (GOx+CAT) and zinc acetate in (1) was added to the mixed solution of hexahistidine-2-methylimidazole-triethylamine (2), stirred at room temperature at 500 rpm for 15 min, and allowed to stand for 8 h.

[0176] (4) The obtained mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain the (GOx+CAT)&4His6@ZIF-8 immobilized enzyme.

[0177] Comparative Example 5

[0178] (1) Dissolve 1 mg CAT and 2 mg GOx in 2 mL 0.04 M zinc acetate solution and stir at 500 rpm for 1 min.

[0179] (2) Mix 2 mL of 0.16 M 2-methylimidazole and 30 μL of triethylamine solution and stir at 500 rpm for 1 min.

[0180] (3) The mixed solution of (GOx+CAT) and zinc acetate in (1) was added to the mixed solution of 2-methylimidazole-triethylamine (2), stirred at 500 rpm for 15 min at room temperature, and allowed to stand for 8 h.

[0181] (4) The obtained mixture was washed three times by centrifugation with high-purity water, and the precipitate was collected to obtain the (GOx+CAT)@ZIF-8 immobilized enzyme.

[0182] Experimental Example 7

[0183] (1) Enzyme activity determination: 4 mg of glucose was taken as substrate solution and mixed with (GOx+CAT)&4His6@ZIF-8 immobilized enzyme solution, (GOx+CAT)@ZIF-8 immobilized enzyme solution and an equal amount of free (GOx+CAT) (500 μL, 1 g GOx, 0.5 g CAT). The oxygen content in the solution was detected by an oxygen meter to obtain the relative activity value of the cascade reaction enzyme. Fig.13 ) As can be seen from the figure, the relative activity of the directly mineralized (GOx+CAT)&4His6@ZIF-8 immobilized enzyme is 284.0%, and its relative enzyme activity is significantly increased to 571.4% after doping with hexahistidine.

[0184] (2) Enzyme stability test: 500 μL of (GOx+CAT)&4His6@ZIF-8, (GOx+CAT)@ZIF-8 and 500 μL of free (GOx+CAT) enzyme were taken and incubated with 500 μL of MeOH, DMSO and CH2Cl2 solvent for 10 min respectively. The dissolved oxygen content of (GOx+CAT)&4His6@ZIF-8, (GOx+CAT)@ZIF-8 and free (GOx+CAT) enzyme after treatment was measured using a dissolved oxygen detector, and the relative enzyme activity was calculated.

[0185] Depend on Fig.15 It can be seen that in an organic solvent environment, the activity of the enzyme (GOx+CAT)&4His6@ZIF-8 can still be retained by more than 420%, while the activity of the (GOx+CAT)@ZIF-8 immobilized enzyme is less than 270%, and the free enzyme retains less than 15% of its activity.

[0186] It can be seen that the present invention immobilizes a variety of enzymes by doping amino acids or polypeptides into porous carriers, which has high enzyme encapsulation efficiency, improved enzyme stability, and improved enzyme catalytic efficiency; not only that, it also improves the recycling performance of the enzyme and can efficiently capture active oxygen in cells.

[0187] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for in situ enzyme immobilization to enhance enzyme activity, characterized in that: The following steps are involved: mixing the oxidoreductase with the metal ion solution to obtain an enzyme-metal ion mixed solution; mixing the nitrogen-containing organic molecule with the ligand to obtain a nitrogen-containing organic molecule-ligand mixed solution; The enzyme-metal ion mixed solution is mixed with the nitrogen-containing organic molecule-ligand mixed solution for immobilization to obtain the in-situ immobilized enzyme.

2. The method according to claim 1, characterized in that: The oxidoreductase comprises at least one of the following enzymes: catalase, glucose oxidase and horseradish peroxidase; The mass volume ratio of the oxidoreductase to the metal ion solution is 0.8-1.2 mg:1 mL.

3. The method according to claim 1, characterized in that The metal ion solution includes the following metal ions: Zn 2+ 、Co 2+ or Ca 2+ ; The concentration of the metal ion solution is 0.04-0.5M.

4. The method according to claim 1, characterized in that: The nitrogen-containing organic molecule includes an amino acid or a polypeptide; The amino acid includes alanine, isoleucine, alanine, cysteine, aspartic acid, glutamic acid, lysine, arginine or histidine; The polypeptide comprises trimeryl histidine, hexahistidine or nonahistidine.

5. The method according to claim 1, characterized in that: The mass volume ratio of the nitrogen-containing organic molecule to the ligand is 1-5 mg:2 mL.

6. The method according to claim 1, characterized in that The ligands include organic ligands or anionic ligands; The organic ligand includes 2-methylimidazole or 2-aldehyde imidazole; the anionic ligand includes sodium carbonate or disodium hydrogen phosphate.

7. The method according to claim 1, characterized in that The concentration of the ligand is 0.1-0.5M.

8. The method according to claim 1, characterized in that The immobilization temperature is 20-30° C.; the immobilization pH is 6-9.

9. An in situ immobilized enzyme obtained by the method according to any one of claims 1 to 8.

10. Use of the in situ immobilized enzyme obtained by the method according to any one of claims 1 to 8 or the in situ immobilized enzyme according to claim 9 in the preparation of a reagent for removing active oxygen from cells.

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