Immobilized enzyme catalyst based on epoxy modified covalent organic framework carrier and preparation method thereof

Abstract

The invention discloses an immobilized enzyme catalyst based on an epoxy modified covalent organic framework (COF) carrier and a preparation method of the immobilized enzyme catalyst, and belongs to the technical field of biological catalysis. The preparation method comprises the following steps: firstly, mixing COF-OH, epoxy chloropropane and a sodium hydroxide solution, stirring and reacting for 6-24 hours to obtain a modified material COF-EP; and dispersing COF-EP in a buffer solution, adding an enzyme solution, carrying out a constant-temperature reaction, collecting a solid product, and drying the solid product to prepare the enzyme # imgabs0 # COF-EP catalyst. By adjusting functional groups of COF, the method provides covalent binding sites for enzyme, and compared with physical adsorption, enzyme conformation change is more effectively induced, active sites are more effectively exposed, and therefore the catalytic performance is improved. The obtained catalyst has high enzyme activity, large enzyme loading capacity and excellent operation stability. The preparation method is low in cost, mild in condition and simple and convenient to operate, can be popularized to other immobilized enzyme systems, and expands the application prospect of the COF material in the field of biological catalysis.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and in particular relates to an immobilized enzyme catalyst based on an epoxy-modified covalent organic framework carrier and a preparation method thereof. Background Art

[0002] Enzymes are a class of natural catalysts with high catalytic activity that can drive intracellular biotransformations under green, mild and efficient conditions. Their unique catalytic efficiency has attracted scientists to explore strategies for applying them to the extracellular environment. In the fields of organic synthesis, drug development, energy conversion, environmental remediation and biomedicine, enzymes show advantages that traditional synthetic catalysts cannot match. The structure of enzymes is dynamically maintained by intramolecular hydrogen bonds, hydrophobic interactions and electrostatic interactions, but these interactions make enzymes thermodynamically unstable and easily affected by external stimuli. In addition, the recycling of enzymes is difficult, resulting in high application costs. Studies have shown that enzyme immobilization is one of the effective ways to deal with these problems. Enzyme immobilization technology attaches enzymes to solid carriers, which not only simplifies the operation process, which can usually be achieved under mild conditions, but also reduces the interference of the external environment on the enzyme, and uses the spatial restriction of the carrier to stabilize the three-dimensional structure of the enzyme. In addition, the mechanical properties of the carrier also contribute to the separation and reuse of the enzyme, providing more possibilities for industrial applications.

[0003] Traditional polymers and inorganic materials have been widely used as carrier materials for many years. However, with the advancement of porous nanomaterial technology, metal organic frameworks (MOFs) and covalent organic frameworks (COFs) have gradually stood out due to their excellent performance. COFs are a class of crystalline porous polymers composed of organic structural units connected by covalent bonds. They have the characteristics of regular structure, low density, large specific surface area and rich active sites. Therefore, they have become the frontier research object in the field of porous nanomaterials. COFs can not only form stable complexes with enzymes, but also their pore size can be customized according to the size of the enzyme. At the same time, their pore structure can optimize the mass transfer efficiency, making them an ideal carrier material for enzyme immobilization. Sachin T. et al. (Chemical Engineering Journal, 2022) used amine ligands such as p-phenylenediamine and 1,3,5-tris(4-aminophenyl)benzene, as well as 2,4,6-trihydroxybenzene-1,3,5-triformaldehyde, to prepare enzyme@COFs catalysts by pre-crosslinking the amine groups on the enzyme surface with the aldehyde groups of COFs. The activity of immobilized glucose oxidase is only 62% of that of the free enzyme. Kang L. et al. (Angewandte Chemie International Edition, 2023) used a small amount of dimethylformamide and acetic acid to synthesize COF-LZU1 by room temperature self-assembly, and the catalytic activity of immobilized horseradish peroxidase was retained at a maximum of 87.6%. He H. et al. (Nature Communications, 2024) used glyoxylic acid to catalyze 2,5-bis(allyloxy)terephthaloylhydrazide and trimesic acid to synthesize NKCOF-141 at room temperature, and pretreated the enzyme with ethylenediaminetetraacetic acid and sodium N-hydroxysuccinimidyl. The final immobilized enzyme catalytic activity was 87% of the free enzyme, and 77% of the initial activity was retained after 6 repeated uses. Although some progress has been made in immobilized enzyme technology, the activity of immobilized enzymes is generally still lower than that of free enzymes, and there is still room for improvement in preventing enzyme leaching and leakage, mainly because the binding between COFs and enzymes mainly relies on relatively weak physical adsorption. Summary of the invention

[0004] In order to overcome the deficiencies in the prior art, the present invention proposes an enzyme immobilization method based on an epoxy-modified covalent organic framework carrier. The method introduces covalent bonding sites into the COFs material so that the enzyme can be stably loaded on the COFs, thereby improving the catalytic performance, loading capacity and stability of the immobilized enzyme. The amino groups on the surface of the enzyme can react with the epoxy groups to form covalent interactions. Compared with physical adsorption, covalent bonding has a stronger interaction force, which can effectively interfere with the interaction within the enzyme molecule, change its natural conformation, and thus increase the exposure of the active site.

[0005] The purpose of the present invention is achieved through the following technical solutions: A method for preparing an immobilized enzyme catalyst based on an epoxy-modified covalent organic framework carrier, characterized in that it comprises the following steps: (1) Preparation of COF-EP: Mix the hydroxy covalent organic framework COF-OH with the epoxy-containing compound, add an alkaline solution for ultrasonic treatment and stir to react for 6-24 hours, collect the solid product, wash, extract and dry to obtain the epoxy-modified material COF-EP; (2) Preparation of enzyme catalyst: Disperse COF-EP in buffer solution, add enzyme solution, react at constant temperature, centrifuge, wash the solid product and dry it to obtain immobilized enzyme catalyst GOx COF-EP.

[0006] Preferably, the amine ligands of the hydroxyl covalent organic framework COF-OH are 2,4,6-tris(4-aminophenyl)-1,3,5-triazine TAPT and / or 1,3,5,-tris(4-aminophenyl)benzene TAPB, and the aldehyde ligands are one or more of trialdehyde pyroglutamic acid TFP, 2-hydroxy-1,3,5-benzenetricarboxaldehyde HBT, 2,4-dihydroxybenzene-1,3,5-tricarboxaldehyde DBT, and 2,5-dihydroxyterephthalaldehyde DHTA.

[0007] Preferably, the hydroxyl covalent organic framework COF-OH is prepared by the following method: adding amine ligands and aldehyde ligands to an acetic acid solution, ultrasonically reacting for 0.5 to 3 hours, and collecting a solid product as COF-OH; the mass ratio of the amine ligand to the aldehyde ligand is (1 to 5): 1, the concentration of the acetic acid solution is 8 to 15 M, and the ultrasonic power is 100 to 500 W.

[0008] Preferably, in step (1), the epoxy-containing compound is epichlorohydrin, and the mass ratio of COF-OH to the epoxy-containing compound is 1:(5-20); and the alkaline solution is 0.5-2 M NaOH.

[0009] Preferably, in step (1), the mass ratio of COF-OH to the epoxy-containing compound is 1:8, the stirring speed is 200-500 r / min, and the temperature is 20-50°C.

[0010] Preferably, in step (1), the washing step is washed with methanol and acetone successively, and the extraction step is carried out with tetrahydrofuran for Soxhlet extraction for 10 to 34 hours, and the drying temperature is 40 to 60° C. and the time is 10 to 15 hours.

[0011] Preferably, in step (2), the enzyme is an oxidoreductase, and the mass ratio of the enzyme to COF-EP is 1:(1-10).

[0012] Preferably, in step (2), the enzyme in step (2) is one or more of glucose oxidase GOx, glucose isomerase GI, lactate oxidase LOx, nonspecific peroxygenase UPO, and cytochrome P450.

[0013] Preferably, in step (2), the isothermal reaction conditions are a rotation speed of 150-250 r / min, a temperature of 20-30°C, and a time of 12-18 hours, and the buffer is a phosphate buffer; the centrifugation parameters are 5000-10000 rpm, and the time is 5-30 minutes, and the washing and centrifugation are performed 1-5 times, and the drying conditions are 30-80°C and the time is 10-14 hours.

[0014] The immobilized enzyme catalyst based on an epoxy-modified covalent organic framework carrier is prepared by the method of the invention.

[0015] The enzyme catalyst provided by the present invention has the following advantages: (1) COFs materials with epoxy functional groups were synthesized through post-modification strategies, and the activity and stability of immobilized enzyme catalysts were significantly improved. The covalently bound immobilization method maintained high catalytic activity in a variety of solvents, which helped to optimize the performance of the enzyme. (2) The raw materials used are simple and easy to obtain, the preparation cost is low, the reaction conditions are mild, and the operation is simple and efficient; (3) The epoxy functional group provides a stable covalent connection, which effectively improves the catalytic performance and stability of the enzyme. At the same time, the COFs carrier can prevent the enzyme from being inactivated under harsh conditions and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the mechanism of modification of COF-OH into COF-EP described in Example 2.

[0017] Figure 2 This is a comparison chart of XRD before and after modification of COF-EP prepared in Example 2 and COF-OH prepared in Comparative Example 1.

[0018] Figure 3 GOx prepared in Example 2 Bar graph of relative enzyme activities of COF-EP catalyst, GOx@COF-OH catalyst prepared in Comparative Example 1, and GOx+COF-EP prepared in Comparative Example 2.

[0019] Figure 4 GOx prepared in Example 2 Comparison of the cyclic stability of COF-EP catalyst and GOx@COF-OH catalyst prepared in Comparative Example 1.

[0020] Figure 5GOx prepared in Example 2 Comparative diagram of the organic solvent tolerance stability of COF-EP catalyst and GOx@COF-OH catalyst prepared in Comparative Example 1. DETAILED DESCRIPTION

[0021] The present invention will be further described in detail through the following specific examples and drawings, but the implementation modes are not limited thereto.

[0022] All methods described in the present invention are conventional technical means unless otherwise specified, and all raw materials can be obtained through public commercial channels unless otherwise specified.

[0023] Example 1 (1) Preparation of COF-EP 150 mg of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine was weighed as an amine ligand, and 89 mg of trialdehyde phloroglucinol was weighed as an aldehyde ligand, and added to a 12 M acetic acid solution. The mixture was ultrasonically reacted at 300 W for 1 hour using a cell disruptor, and the solid product was collected to obtain COF-OH. Then 60 mg of COF-OH and 500 mg of epichlorohydrin were weighed, added to a 100 mL beaker and mixed, and then 40 mL of 1.5 M NaOH solution was added. After ultrasonic treatment, the mixture was stirred at 40 °C for 8 hours, and the solid product was collected and washed with methanol and acetone, then extracted with tetrahydrofuran Soxhlet for 12 hours, and dried at 50 °C for 12 hours to obtain an epoxy-modified COF material named COF-EP.

[0024] (2) GOx Preparation of COF-EP 25 mg COF-EP was dispersed in 10 mL 10 mM phosphate buffer solution and dispersed evenly by ultrasound. Then 5 mg glucose oxidase (GOx) was added to make the concentrations of COF-EP and GOx reach 2.5 mg / mL and 0.5 mg / mL, respectively. After mixing evenly, it was shaken on a thermostatic shaker for 16 hours (250 rpm, 25°C), and the supernatant was removed by centrifugation (7000 rpm, 10 minutes), washed with phosphate buffer and centrifuged twice, and the solid product was collected and dried at 50°C for 12 hours to obtain GOx. COF-EP catalyst.

[0025] Example 2 (1) Preparation of COF-EP The preparation steps of COF-EP in this example are the same as those in Example 1, except that the stirring time is 12 hours.

[0026] (2) GOx Preparation of COF-EP The enzyme immobilization procedure was the same as in Example 1.

[0027] Example 3 (1) Preparation of COF-EP 150 mg of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine was weighed as an amine ligand, and 89 mg of 2-hydroxy-1,3,5-benzenetricarboxaldehyde was weighed as an aldehyde ligand, and added to a 12 M acetic acid solution. A cell disruptor was used for ultrasonic reaction at a power of 300 W for 1 hour, and the solid product was collected to obtain COF-OH. The subsequent steps were the same as in Example 1, and the stirring time was 12 hours.

[0028] (2) GOx Preparation of COF-EP The enzyme immobilization procedure was the same as in Example 1.

[0029] Comparative Example 1 150 mg of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine and 89 mg of trialdehyde phloroglucinol were weighed and added to 12M acetic acid solution. The solid product was collected by using a cell disruptor at 300 W power for 1 hour to obtain COF-OH. 25 mg of COF-OH was dispersed in 10 mL of phosphate buffer solution. After ultrasonic dispersion, 5 mg of glucose oxidase (GOx) was added. After mixing, it was shaken on a constant temperature shaker for 16 hours, centrifuged (7000 rpm, 10 minutes), the supernatant was removed, washed with phosphate buffer and centrifuged twice, the solid was collected and dried to obtain GOx@COF-OH catalyst.

[0030] Comparative Example 2 The steps for preparing COF-EP were the same as those in Example 3. Subsequently, 25 mg of COF-EP was dispersed in 10 mL of phosphate buffer solution, and after ultrasonic dispersion, 5 mg of glucose oxidase (GOx) was added and mixed to obtain GOx+COF-EP catalyst.

[0031] Example Effect Description Figure 2 The XRD patterns of the COF-OH and COF-EP materials described in Examples 2 and 3 and Comparative Examples 1 and 2, wherein the reaction solvent conditions used in the modification of Examples 2 and 3 and Comparative Example 2 are 60 mg COF-OH, 500 mg epichlorohydrin, 40 mL 1.5 M NaOH solution, and stirring for 16 h.

[0032] Figure 2The XRD graphs shown show that the original crystal structures of the COF-OH and COF-EP materials in Examples 2 and 3 and Comparative Examples 1 and 2 were not damaged during the epoxide modification process. The XRD diffraction characteristic peaks (5.6°, 9.8°, 25.3°) of the COFs material remained consistent before and after the modification, indicating that the crystallinity of the material did not change significantly.

[0033] Table 1 shows the GOx prepared in Examples 1 to 3 and Comparative Example 1 COF-EP, GOx@COF-OH, and GOx prepared in Example 2 and Comparative Example 2 Comparison of enzyme loading rate and relative enzyme activity of COF-EP and GOx+COF-EP catalysts. The ligand column indicates the number of hydroxyl groups contained in the aldehyde ligand of the COF material used. The aldehyde ligands corresponding to Examples 1 and 2 and Comparative Examples 1 and 2 contain three hydroxyl groups, and the aldehyde ligand corresponding to Example 3 contains one hydroxyl group. Among them, all COFs in the examples are modified by epoxy group. GOx When COF-EP, GOx@COF-OH and GOx+COF-EP were compared with GOx in the free state, the activity of the free enzyme was taken as 100%.

[0034] Enzyme loading: The BCA method was used to test the difference in enzyme concentration in the supernatant before and after loading, so as to calculate the enzyme loading in COFs. According to the BCA method, 20 μL of sample was added to a 96-well plate, and then 200 μL of BCA working solution was added. After reacting in an incubator at 37 °C and 200 rpm for 30 min, the sample was detected by UV-visible spectrophotometer. The protein concentration is proportional to the absorption intensity at 562 nm. The enzyme loading was calculated using formula (1-1).

[0035] In formula (1-1), C1 (mg / mL) is the initial concentration of the enzyme solution added, V1 (mL) is the volume of the enzyme solution added, C2 (mg / mL) is the concentration of the enzyme in the supernatant, V2 (mL) is the volume of the supernatant, and when calculating the enzyme loading, m (mg) is the total mass of the carrier sample before fixation, and the unit of loading is mg / g.

[0036] Enzyme activity test: GOx catalyzes glucose to produce glucose lactone and H2O2, and HRP catalyzes H2O2 to oxidize colorless ABTS into light green ABTS ·+ oxidation state, the ABTS ·+The oxidized state shows a characteristic UV absorption peak at 405 nm. The specific test conditions are as follows: the reaction is carried out in 1 mL of PBS buffer (10 mM, pH 7.0), in which the ABTS concentration is 2.5 mM, the glucose concentration is 2.5 mM, the GOx concentration is 10 nM, and the HRP concentration is 500 nM. The enzyme content is equal in different samples participating in the reaction. The reaction is carried out at room temperature, and the absorbance of the solution at 405 nm is recorded when the reaction lasts for 1 min, and compared with the free enzyme to calculate the enzyme activity retention.

[0037] The results in Table 1 show that GOx The enzyme loading of COF-EP was significantly higher than that of GOx@COF-OH, especially in Example 2. The loading amount of COF-EP reached 40.2 mg / g, and the relative enzyme activity was 268.2%, showing the best catalytic performance, which is much higher than the activity of free enzyme. By comparing Examples 1 and 2, it can be seen that as the COF modification time increases, the modification degree increases, and the corresponding enzyme activity also increases significantly. In contrast, Example 3 has the same modification time as Example 2, but because the aldehyde ligand used in Example 3 contains a small number of hydroxyl groups, its relative enzyme activity is ultimately lower than that of Example 2. This shows that the modification degree and the number of hydroxyl groups of COF have an important influence on the immobilization effect of the enzyme.

[0038] In Comparative Example 2, the relative enzyme activity of the catalyst (GOx+COF-EP) in which GOx and COF-EP were directly mixed without being treated with a constant temperature shaker was only 103.1%, which was almost the same as the activity of the free enzyme, and was significantly lower than that of Example 2 of covalent immobilization and Comparative Example 1 of physical adsorption. This shows that the interaction between the enzyme and COF plays a key role in the enzyme activity, especially the covalent immobilization method can significantly enhance the catalytic performance of the enzyme. In summary, the conditions in Example 2 with a mass ratio of COF-OH to epichlorohydrin of 1:8, an aldehyde ligand containing three hydroxyl groups, a NaOH concentration of 1.5 M, and a modification reaction time of 16 hours were proven to be the optimal solution for preparing COF-EP. These conditions achieve the best balance between enzyme loading and enzyme activity, significantly improving the catalytic effect of the immobilized enzyme.

[0039] Table 1 Enzyme loading and relative enzyme activity of enzyme@COFs catalysts a: -3OH means that the COF aldehyde monomer is trialdehyde phloroglucinol, which contains three hydroxyl groups; -OH means that the COF aldehyde monomer is 2-hydroxy-1,3,5-benzenetricarboxaldehyde, which contains one hydroxyl group.

[0040] Figure 3 The GOx prepared in Example 2 is shown The relative enzyme activity bar graph of COF-EP, GOx@COF-OH of Comparative Example 1, and GOx+COF-EP of Comparative Example 2. The experimental results show that GOx The relative activity of COF-EP catalyst was significantly increased from 216.4% before modification to 268.2%. Compared with GOx+COF-EP without covalent fixation (relative activity of 103.1%), GOx COF-EP showed a significant increase in activity, indicating that covalent immobilization can effectively enhance the interaction between the enzyme and the COF carrier, thereby improving the catalytic performance of the enzyme. In addition, the enzyme reaction kinetics data showed that GOx The KM value of COF-EP is 10.3 mM, which is significantly lower than that of free GOx (20.3 mM), indicating that the covalently fixed enzyme has a higher affinity for the substrate. It is speculated that this is because the structure of COFs induces conformational changes in the enzyme, making the active center of the enzyme more exposed and promoting the contact between the substrate and the enzyme active site.

[0041] Figure 4 The GOx of Example 2 is shown Activity changes of COF-EP and GOx@COF-OH of Comparative Example 1 after 6 consecutive reaction cycles. COF-EP still maintains more than 95% of its initial activity after 6 cycles, which is significantly better than 65% of GOx@COF-OH, indicating that GOx COF-EP has good reusability and stability after multiple uses.

[0042] Figure 5 The changes in enzyme activity after 12 hours of treatment with different organic solvents are shown. The activity of free GOx in tetrahydrofuran decreased significantly, retaining only 61.2% of the initial activity, while the activity also decreased to varying degrees in other solvents such as acetone, ethanol, and dimethyl sulfoxide. In contrast, the covalently fixed GOx The activity of COF-EP in acetone, ethanol, and dimethyl sulfoxide remained above 90%, and the activity retention in tetrahydrofuran reached 84.5%. In contrast, the activity retention of physically adsorbed GOx@COF-OH in these solvents was lower. These results indicate that GOx COF-EP provides better structural protection through covalent fixation and reduces the interference of organic solvents on the enzyme conformation, thereby improving the solvent resistance and stability of the enzyme.

[0043] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing an immobilized enzyme catalyst based on an epoxy-modified covalent organic framework carrier, characterized in that: The following steps are involved: (1) Preparation of COF-EP: Mix the hydroxy covalent organic framework COF-OH with the epoxy-containing compound, add an alkaline solution for ultrasonic treatment and stir to react for 6-24 hours, collect the solid product, wash, extract and dry to obtain the epoxy-modified material COF-EP; (2) Preparation of enzyme catalyst: Disperse COF-EP in buffer solution, add enzyme solution, react at constant temperature, centrifuge, wash the solid product and dry it to obtain immobilized enzyme catalyst GOx COF-EP.

2. The method for preparing an immobilized enzyme catalyst based on an epoxy-modified covalent organic framework carrier according to claim 1, characterized in that: The amine ligands of the hydroxy covalent organic framework COF-OH are 2,4,6-tris(4-aminophenyl)-1,3,5-triazine TAPT and / or 1,3,5,-tris(4-aminophenyl)benzene TAPB, and the aldehyde ligands are one or more of trialdehyde phloroglucinol TFP, 2-hydroxy-1,3,5-benzenetricarbaldehyde HBT, 2,4-dihydroxybenzene-1,3,5-tricarbaldehyde DBT, and 2,5-dihydroxyterephthalaldehyde DHTA.

3. The method for preparing an immobilized enzyme catalyst based on an epoxy-modified covalent organic framework carrier according to claim 2, characterized in that: The hydroxyl covalent organic framework COF-OH is prepared by the following method: adding amine ligands and aldehyde ligands to an acetic acid solution, ultrasonically reacting for 0.5 to 3 hours, and collecting a solid product as COF-OH; the mass ratio of the amine ligand to the aldehyde ligand is (1 to 5): 1, the concentration of the acetic acid solution is 8 to 15M, and the ultrasonic power is 100 to 500W.

4. The method for preparing an immobilized enzyme catalyst based on an epoxy-modified covalent organic framework carrier according to any one of claims 1 to 3, characterized in that: In step (1), the epoxy-containing compound is epichlorohydrin, and the mass ratio of COF-OH to the epoxy-containing compound is 1:(5-20); and the alkaline solution is 0.5-2 M NaOH.

5. The method for preparing an immobilized enzyme catalyst based on an epoxy-modified covalent organic framework carrier according to claim 4, characterized in that: In step (1), the mass ratio of the COF-OH to the epoxy-containing compound is 1:8, the stirring speed is 200-500 r / min, and the temperature is 20-50°C.

6. The method for preparing an immobilized enzyme catalyst based on an epoxy-modified covalent organic framework carrier according to claim 5, characterized in that: In step (1), the washing step is performed with methanol and acetone in sequence, and the extraction step is performed with tetrahydrofuran for Soxhlet extraction for 10 to 34 hours, with a drying temperature of 40 to 60° C. and a drying time of 10 to 15 hours.

7. The method for preparing an immobilized enzyme catalyst based on an epoxy-modified covalent organic framework carrier according to claim 6, characterized in that: In step (2), the enzyme is an oxidoreductase, and the mass ratio of the enzyme to COF-EP is 1:(1-10).

8. The method for preparing an immobilized enzyme catalyst based on an epoxy-modified covalent organic framework carrier according to claim 7, characterized in that: In step (2), the enzyme in step (2) is one or more of glucose oxidase GOx, glucose isomerase GI, lactate oxidase LOx, non-specific peroxygenase UPO, and cytochrome P450.

9. The method for preparing an immobilized enzyme catalyst based on an epoxy-modified covalent organic framework carrier according to claim 8, characterized in that: In step (2), the constant temperature reaction conditions are a rotation speed of 150-250 r / min, a temperature of 20-30°C, and a time of 12-18 hours, and the buffer is a phosphate buffer; the centrifugation parameters are 5000-10000 rpm, the time is 5-30 minutes, washing and centrifugation are performed 1-5 times, and the drying conditions are 30-80°C and the time is 10-14 hours.

10. An immobilized enzyme catalyst based on an epoxy-modified covalent organic framework carrier prepared by the method of any one of claims 1 to 9.

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