Covalent organic framework material, method of making the same, hybrid organelle made of the covalent organic framework material, method of making the hybrid organelle, and applications

Abstract

This application discloses a covalent organic framework material and its preparation method, hybrid organelles prepared from the covalent organic framework material, the preparation method, and its applications, relating to the field of bioenzyme engineering. The preparation method of the covalent organic framework material includes: dissolving a monomer containing an acylhydrazine functional group and 2,5-bis(2-methoxyethoxy)terephthalohydrazide containing polyethylene glycol in an aqueous organic solvent to form a dispersion; adding a monomer solution containing an aldehyde functional group to the dispersion; carrying out an immobilization reaction under catalytic conditions; and obtaining the covalent organic framework material after centrifugation and purification. Thus, this application introduces a PEG-driven emulsion template method to prepare the covalent organic framework material COFcap-2. This covalent organic framework material can immobilize various biological elements, improve their tolerance to adverse environments, and achieve a protective function.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological enzyme engineering, and particularly relates to a covalent organic framework material, a preparation method thereof, a hybrid organelle prepared from the covalent organic framework material, a preparation method and application thereof. BACKGROUND

[0002] Taking CO2 and N2 as raw materials and taking clean energy such as electric energy and light energy to synthesize substances is an important path for saving fossil resources, protecting the environment and realizing green manufacturing, and is also a focus of international scientific and technological competition. Existing manufacturing routes are mostly concentrated in the reduction synthesis of organic one-carbon / one-nitrogen molecules from CO2 and N2. However, multi-carbon / multi-nitrogen compounds have more extensive applications in the fields of biological medicine, pesticides, fine chemicals and material science, and therefore, the manufacturing route for the reduction synthesis of high-value-added multi-carbon / multi-nitrogen compounds from CO2 and N2 needs to be broken through.

[0003] At present, a single biological element or non-biological element cannot realize such a complex catalytic process, and the manufacturing of multi-carbon / multi-nitrogen compounds can be completed through a composite catalytic system reasonably integrating multiple elements. However, a homogeneous catalytic system often faces problems such as low mass transfer rate, low conversion efficiency, poor stability and difficulty in recycling. SUMMARY

[0004] The application provides a covalent organic framework material, a preparation method thereof, a hybrid organelle prepared from the covalent organic framework material, a preparation method and application thereof, so as to overcome the technical problems of low mass transfer rate, low conversion efficiency, poor stability and difficulty in recycling in the existing homogeneous catalytic system, and effectively realize the immobilization of multiple biological elements and improve the tolerance of the biological elements to adverse environments. Meanwhile, through a self-assembly strategy, the co-immobilization of multiple biological elements (enzymes) and non-biological elements (nanoparticles and coenzyme factors) is realized.

[0005] In order to achieve the above-mentioned purpose, the technical scheme of the embodiment of the application is as follows:

[0006] The first aspect of the application provides a preparation method of a covalent organic framework material, comprising the following steps:

[0007] A monomer containing a hydrazide group functional group and a 2,5-bis(2-methoxyethoxy) terephthalic dihydrazide containing polyethylene glycol are dissolved in an organic solvent aqueous solution to form a dispersion liquid;

[0008] A monomer solution containing an aldehyde group functional group is added to the dispersion liquid, and a immobilization reaction is carried out under the condition of a catalyst. After centrifugal purification, the covalent organic framework material is obtained.

[0009] Preferably in combination with the first aspect, the monomer containing a hydrazide group functional group is at least one of 2,5-diethoxybenzene-1,4-di(formhydrazide), 2,5-bis(2-methoxyethoxy)terephthalic dihydrazide, 2,5-bis(but-3-en-1-yloxy)terephthalic dihydrazide, 2-(but-3-en-1-yloxy)-5-(2-methoxyethoxy)terephthalic dihydrazide;

[0010] Preferably in combination with the first aspect, the polyethylene glycol in the 2,5-bis(2-methoxyethoxy)terephthalic dihydrazide containing polyethylene glycol is at least one of 400 g / mol, 600 g / mol, 800 g / mol;

[0011] Preferably in combination with the first aspect, the monomer containing an aldehyde group functional group is at least one of trimesic aldehyde, 2,6-dihydroxy-1,3,5-benzene tricarboxaldehyde, tricarbonyl m-phenol;

[0012] Preferably in combination with the first aspect, the catalyst is at least one of a compound containing anhydride functional group, a compound containing carboxylic acid functional group, a compound containing imidazole functional group, a compound containing hydroxyl functional group.

[0013] Preferably in combination with the first aspect, the molar ratio of the monomer containing a hydrazide group functional group, the 2,5-bis(2-methoxyethoxy)terephthalic dihydrazide containing polyethylene glycol, and the monomer containing an aldehyde group functional group is 0:30:20, 1:29:20, 3:27:20, 5:25:20, 10:20:20, 20:10:20, 30:0:20;

[0014] Preferably in combination with the first aspect, the volume ratio of water and organic solvent in the aqueous organic solvent solution is 0.5:7.5, 1.0:7.5, 2.0:7.5, 3.0:7.5.

[0015] Preferably in combination with the first aspect, the molar ratio of the monomer containing a hydrazide group functional group, the 2,5-bis(2-methoxyethoxy)terephthalic dihydrazide containing polyethylene glycol, and the monomer containing an aldehyde group functional group is 3:27:20;

[0016] Preferably in combination with the first aspect, the volume ratio of water and organic solvent in the aqueous organic solvent solution is 1.0:7.5;

[0017] Preferably in combination with the first aspect, the catalyst is at least one of acetic acid, scandium triflate.

[0018] The second aspect of the present application provides a use of the covalent organic framework material prepared by any of the methods of the first aspect for preparing a covalent organic framework hybrid organelle.

[0019] The third aspect of the present application provides a preparation method of the covalent organic framework hybrid organelle of the second aspect, the method comprising:

[0020] dissolving the monomer containing an aldehyde functional group and the monomer containing a hydrazine functional group in an organic solvent aqueous solution to form a dispersion;

[0021] resuspending and emulsifying the nanoparticles, the coenzyme factor and the biological enzyme in an aqueous solution, adding a monomer solution containing an aldehyde functional group, and performing an immobilization reaction under the condition of scandium triflate as a catalyst, and then centrifuging and purifying to obtain the covalent organic framework hybrid organelle.

[0022] In combination with the third aspect, preferably, the monomer containing an aldehyde functional group is at least one of triformylphloroglucinol, 2,6-dihydroxy-1,3,5-benzene triformaldehyde, and triformylphloroglucinol;

[0023] and / or, at least one of 2,5-bis(2-methoxyethoxy)terephthalic hydrazide, 2,5-bis(but-3-en-1-oxy)terephthalic hydrazide, and 2-(but-3-en-1-oxy)-5-(2-methoxyethoxy)terephthalic hydrazide;

[0024] and / or, the organic solvent is chloroform;

[0025] and / or, the nanoparticles are at least one of gold nanoparticles, iron nanoparticles, and copper nanoparticles;

[0026] and / or, the biological enzyme is at least one of transaminase, glucose dehydrogenase, glutamate dehydrogenase, leucine dehydrogenase, glucose oxidase, catalase, and lipase.

[0027] In combination with the third aspect, preferably, the molar ratio of the monomer containing an aldehyde functional group to the monomer containing a hydrazine functional group is 2:3;

[0028] and / or, the temperature of the immobilization reaction is 10-40°C;

[0029] and / or, the addition amount of the biological enzyme is 0.25-1.0 mg;

[0030] and / or, the addition amount of scandium triflate is 10-70 μL;

[0031] and / or, the addition amount of the coenzyme factor is 5.0 mg.

[0032] In combination with the third aspect, preferably, the addition amount of the biological enzyme is 0.5 mg;

[0033] and / or, the temperature of the immobilization reaction is 20-30°C.

[0034] The fourth aspect of the present application provides a use of the covalent organic framework hybrid organelle prepared by the method of the third aspect for producing a pharmaceutically active ingredient or a pharmaceutical intermediate.

[0035] Compared with the prior art, the advantages or beneficial effects of the embodiments of the present application at least include:

[0036] The present application introduces a PEG-driven emulsion template method to prepare a covalent organic framework material COFcap-2, which can fix various biological elements and improve their tolerance to adverse environments to achieve protection.

[0037] The present application can use the covalent organic framework material COFcap-2 to co-immobilize various enzymes, nanoparticles and coenzyme factors, and the prepared covalent organic framework hybrid organelle can catalyze the generation of various chiral amines by electric enzymes, which has higher catalytic efficiency and more excellent stability and recycling than pure enzyme catalytic systems (nitrogenase, transaminase, myocardial yellow enzyme, L-alanine dehydrogenase). The covalent organic framework co-immobilized biological and non-biological element hybrid organelle can shorten the spatial distance because various elements are co-immobilized inside the hybrid organelle, which can effectively promote cascade catalytic reactions, achieve efficient use of energy, and achieve efficient conversion from cheap energy to high-value-added products. In addition, the enzyme tolerance to adverse environments is effectively improved to achieve protection, and excellent recycling is also achieved.

[0038] The present application co-immobilizes various enzymes, nanoparticles and coenzyme factors through a self-assembly strategy. Various enzymes and various chemical catalysts are mixed with monomer 1 containing a hydrazide group functional group and monomer containing an aldehyde group functional group in a reaction solvent, and a condensation reaction is carried out under the condition of scandium triflate as a catalyst, and centrifugal washing to obtain co-immobilized enzymes-chemical catalysts. The hybrid organelle co-immobilized biological and non-biological elements includes but is not limited to application in the medical field of producing pharmaceutically active ingredients or pharmaceutical intermediates, and has excellent catalytic activity, stability and recycling. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is a schematic diagram of the structure of the covalent organic framework material COFs provided by the present application;

[0040] Figure 2 is a transmission electron microscope image (a), a powder X-ray diffraction image (b), a gas adsorption image (c) and a shell thickness schematic diagram (d) of COFcap-2 formed by different monomer ratios;

[0041] Figure 3 is a TEM image (a) and a dynamic light scattering image (b) of COFcap-2 formed by different solvent ratios;

[0042] Figure 4 is a transmission electron microscope image of the product prepared in Example 3;

[0043] Figure 5 is an X-ray photoelectron spectroscopy image (a) and NAD + leakage image (b) of the product prepared in Example 4;

[0044] Figure 6 is the enzyme loading (a) and enzyme activity (b) of Lipase@COFcap-2 formed with different monomer ratios;

[0045] Figure 7 is the enzyme loading (a) and enzyme activity (b) of GluDH@COFcap-2;

[0046] Figure 8 is the enzyme loading (a) and enzyme activity (b) of GCDH@COFcap-2;

[0047] Figure 9 is the enzyme loading (a) and enzyme activity (b) of ω-TA@COFcap-2;

[0048] Figure 10 is a laser confocal microscope image of the co-immobilization of GluDH, GCDH and ω-TA by COFcap-2;

[0049] Figure 11 is the ammonia production performance of Au@COFcap-2;

[0050] Figure 12 is the performance of catalytic conversion of nitrogen to chiral amines of catalyst@COFcap-2;

[0051] Figure 13 is the performance of catalytic conversion of nitrogen to chiral amines of catalyst@COFcap-2 formed with different ratios of biocatalysts;

[0052] Figure 14 is the performance of catalytic conversion of nitrogen to different chiral amines of catalyst@COFcap-2;

[0053] Figure 15 is the recyclability of catalyst@COFcap-2. DETAILED DESCRIPTION

[0054] In order to make the purposes, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings, and the described embodiments should not be regarded as limiting the present application. All other embodiments obtained by those of ordinary skill in the art without making creative labor fall within the scope of protection of the present application.

[0055] In the following description, reference is made to "some embodiments", which describe but a subset of all possible embodiments, and it is understood that "some embodiments" can be the same subset or a different subset and can be combined with each other, without conflict, unless otherwise defined. Unless otherwise defined, all technical and scientific terms used in the application embodiments have the same meaning as commonly understood by one of ordinary skill in the art to which the application embodiments belong. The terminology used in the application embodiments is for the purpose of describing the application embodiments only and is not intended to be limiting of the application.

[0056] In the following description of the application embodiments, the terms "including", "containing", "having" and "including" and the like are open-ended terms, i.e., meaning including but not limited to.

[0057] It should be noted that all raw materials / reagents in the application embodiments can be purchased on the market or prepared according to conventional methods well known to those skilled in the art; the term "and / or" in the application embodiments is only used to describe the association relationship of the associated objects, which means that there can be three relationships, for example, A and / or B means that there are three cases of A alone, B alone, and A and B together, wherein A and B can be singular or plural, and the character " / " generally represents an "or" relationship between the associated objects before and after it.

[0058] In the following description of the application embodiments, the term "at least one" means one or more, and "multiple" means two or more. "At least one of the following" or similar expressions means any combination of the items, including any combination of single item or multiple items. For example, "at least one of a, b or c", or "at least one of a, b and c", can mean a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, and c can be single or multiple.

[0059] Those skilled in the art should understand that in the following description of the application embodiments, the order of the serial numbers does not mean the order of execution, and some or all steps can be executed in parallel or in sequence, and the execution order of each process should be determined according to its function and inherent logic, and should not constitute any limitation on the implementation process of the application embodiments.

[0060] The terms used in the application embodiments are only for the purpose of describing the specific embodiments, and are not intended to limit the application. The singular forms "a" and "the" used in the application embodiments and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0061] It should be understood by those skilled in the art that the numerical ranges in the embodiments of the present application should be understood as also specifically disclosing each intermediate value between the upper limit and the lower limit of the range. Each intermediate value between any stated value and stated range, as well as any other stated value or intermediate value in the stated range, is also included in the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently.

[0062] Unless otherwise defined, technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice of the embodiments or test examples of this application. All documents mentioned in this specification are generally cited to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document cited herein, the content of the specification prevails.

[0063] It should be noted that all raw materials and / or reagents in the embodiments of the present application are purchased on the market or prepared according to conventional methods well known to those skilled in the art.

[0064] The present application provides a new method for nitrogen fixation by co-immobilizing enzymes and chemical catalysts in covalent organic framework (COF) capsules (COFcaps). By combining the properties of enzyme molecules and chemical catalysts, effective assembly of enzyme molecules and chemical catalysts under mild conditions is explored and realized, which not only maintains the activity of enzyme molecules and chemical catalysts, but also effectively improves the tolerance to harsh conditions such as organic solvents, proteases, high temperature, etc. The resulting bioelectrocatalytic system is heterogeneous, which can effectively convert N2 into chiral amines and is easy to recover. In addition, the present application not only realizes the immobilization of COFs for various enzyme molecules and chemical catalysts under mild conditions, but also develops a bio-non-bio composite catalytic system with material and energy flow efficiency, and simultaneously uses process engineering integration to shift from the development of single technology / strategy to platform construction.

[0065] In a first aspect, the present application provides a preparation method of a covalent organic framework material, which comprises:

[0066] S10, dissolving a monomer containing a hydrazine group functional group and a 2,5-bis(2-methoxyethoxy) terephthalic hydrazide containing polyethylene glycol in an organic solvent aqueous solution to form a dispersion.

[0067] S20, adding a monomer solution containing an aldehyde group functional group to the dispersion, and performing an immobilization reaction under catalyst conditions, and then centrifuging and purifying to obtain a covalent organic framework material.

[0068] In the present application, a method for synthesizing a plurality of elements using a covalent organic framework material (COFs) is provided. The covalent organic framework material is a covalent organic framework material connected by an imide bond, an imine bond, or a hydrazone bond. The enzyme is a transaminase, glucose dehydrogenase, leucine dehydrogenase, glutamate dehydrogenase, glucose oxidase, catalase, or lipase. The nanoparticle is a gold nanoparticle, iron nanoparticle, or copper nanoparticle. The coenzyme factor is NAD + and NADH.

[0069] This strategy uses a covalent organic framework material to immobilize a plurality of biological elements (enzymes) to improve their tolerance to adverse environments and achieve protection.

[0070] It should be noted that dissolving the monomer containing a hydrazine group functional group and the 2,5-bis(2-methoxyethoxy) terephthaldehyde containing polyethylene glycol in an organic solvent aqueous solution is to mix the raw material components uniformly to form a stable solution dispersion system. Therefore, as long as the mixing method can mix the raw material components uniformly, such as but not limited to stirring, ultrasonic, etc. The conditions such as temperature and time are not particularly required, as long as the dissolution and mixing efficiency of the components such as the monomer containing the hydrazine group functional group, the 2,5-bis(2-methoxyethoxy) terephthaldehyde containing polyethylene glycol, etc. can be considered and implemented.

[0071] In some embodiments, the monomer containing a hydrazine group functional group in the above step S10 is preferably one of 2,5-diethoxybenzene-1,4-di(formylhydrazine), 2,5-bis(2-methoxyethoxy) terephthaldehyde, 2,5-bis(but-3-en-1-oxy) terephthaldehyde, and 2-(but-3-en-1-oxy)-5-(2-methoxyethoxy) terephthaldehyde.

[0072] It should be noted that the molecular weight of the polyethylene glycol in the 2,5-bis(2-methoxyethoxy) terephthaldehyde containing polyethylene glycol in the above step S10 is 400 g / mol, 600 g / mol, or 800 g / mol.

[0073] It should be noted that the organic solvent in the above step S10 is preferably chloroform or acetonitrile.

[0074] It should be noted that the hydrazine group functional group is a functional group containing an acyl group (R-C=O) and a hydrazine group (NH-NH2), and the hydrazine group can also be understood as containing an amino group.

[0075] In some embodiments, the step S20 can be performed by adding the monomer solution containing the aldehyde group functional group into the dispersion solution for thorough reaction, and then performing the immobilization reaction under the catalyst condition, and then performing the centrifugal purification to obtain the covalent organic framework material.

[0076] In some embodiments, the step S20 is performed under the catalyst condition to accelerate the reaction rate and improve the crystallinity of the covalent organic framework material. Therefore, as long as the catalyst can make the monomer solution containing the aldehyde group functional group and the dispersant fully perform the immobilization reaction, the catalyst is within the scope disclosed in the embodiments of the present application, for example, the catalyst can be acetic acid or scandium triflate.

[0077] In some embodiments, the step S20 has no specific implementation method for the purification, for example, the reaction product after the immobilization reaction can be washed, centrifuged, dried and the like. The reaction product can be washed by using deionized water, ethanol, methanol and the like; the drying treatment can be performed at high temperature or in a frozen environment.

[0078] In some embodiments, the monomer containing the aldehyde group functional group in the embodiments of the present application is preferably one of triformylphloroglucinol, 2,6-dihydroxy-1,3,5-benzene tricarboxaldehyde, and tri-aldehyde-based resorcinol.

[0079] It should be noted that the monomer containing the aldehyde group functional group is used as a node monomer in the preparation of the COF framework material, reacts with the monomer containing the amino group to form a network structure connected by covalent bonds. The grid structure provides a stable skeleton and permanent porosity for the COF material. At the same time, the monomer containing the aldehyde group functional group reacts with the monomer containing the amino group functional group to form an imine bond (C=N) or other stable covalent bond during the preparation process. The formation of these covalent bonds guarantees the stability and durability of the COF material.

[0080] In some embodiments, the molar ratio of the monomer containing the hydrazine group functional group, the 2,5-bis(2-methoxyethoxy) terephthalic dihydrazide containing polyethylene glycol, and the monomer containing the aldehyde group functional group is preferably 0:30:20, 1:29:20, 3:27:20, 5:25:20, 10:20:20, 20:10:20, 30:0:20; further preferably, 3:27:20. The molar ratio of different monomers determines whether a hollow capsule is formed or affects the smoothness of the shell and the pore size of the capsule, which affects the substrate transport rate in the catalytic reaction.

[0081] In some embodiments, the volume ratio of water and organic solvent in the organic solvent aqueous solution is preferably 0.5:7.5, 1.0:7.5, 2.0:7.5, 3.0:7.5; further, more preferably 1.0:7.5, and different volume ratios make the capsules have different sizes, determining whether they can be used for immobilizing large-sized nanoparticles, while too large-sized capsules affect the substrate transport rate and affect the catalytic performance.

[0082] In some embodiments, the catalyst is preferably one of acetic acid and scandium triflate.

[0083] In a second aspect, the embodiments of the present application provide a use of the covalent organic framework material prepared by the method of any one of the first aspect for preparing a covalent organic framework hybrid organelle.

[0084] In a third aspect, the embodiments of the present application provide a preparation method of the covalent organic framework hybrid organelle of the second aspect, which comprises:

[0085] S30, dissolving the monomer containing an aldehyde group functional group and the monomer containing a hydrazine group functional group in an organic solvent aqueous solution to form a dispersion;

[0086] S40, resuspending and emulsifying the nanoparticles, coenzyme factors and biological enzymes in an aqueous solution, adding a monomer solution containing an aldehyde group functional group, and performing an immobilization reaction under the condition of scandium triflate as a catalyst, and then centrifuging and purifying to obtain the covalent organic framework hybrid organelle.

[0087] The present application can utilize the covalent organic framework material COFcap-2 to co-immobilize various enzymes, nanoparticles and coenzyme factors, and prepare a covalent organic framework hybrid organelle, which catalyzes the generation of various chiral amines by electric enzymes, has higher catalytic efficiency, more excellent stability and recycling property than a pure enzyme catalytic system (nitrogenase, transaminase, heart muscle yellow enzyme, L-alanine dehydrogenase). The covalent organic framework co-immobilized biological and non-biological element hybrid organelle can shorten the spatial distance because various elements are co-immobilized inside the hybrid organelle, can effectively promote the cascade catalytic reaction, and realize efficient use of energy and efficient conversion from cheap energy to high value-added products. In addition, the enzyme tolerance to poor environment is effectively improved, the protection effect is realized, and the recycling property is excellent.

[0088] It should be noted that the monomer containing an aldehyde group and the monomer containing a hydrazine group are dissolved in an aqueous organic solvent in order to uniformly mix the raw material components and form a stable solution dispersion system. Therefore, as long as the mixing method can sufficiently and uniformly mix the raw material components, any mixing method such as stirring, ultrasonic treatment, and the like can be used. The conditions for dissolution such as temperature and time are not particularly limited, and any conditions that can improve the dissolution and mixing efficiency of the components such as the monomer containing an aldehyde group and the monomer containing a hydrazine group can be used.

[0089] In some embodiments, the monomer containing an aldehyde group in the above step S30 is preferably one of benzene tricarboxaldehyde, 2,6-dihydroxy-1,3,5-benzene tricarboxaldehyde, and tricarboxaldehyde m-hydroxybenzene.

[0090] It should be noted that the monomer containing a hydrazine group in the above step S40 is preferably one of 2,5-bis(2-methoxyethoxy)terephthalic dihydrazide, 2,5-bis(but-3-en-1-oxy)terephthalic dihydrazide, and 2-(but-3-en-1-oxy)-5-(2-methoxyethoxy)terephthalic dihydrazide.

[0091] It should be noted that the organic solvent in the above step S30 is preferably chloroform.

[0092] It should be noted that the hydrazine group is a functional group containing an acyl group (R-C=O) and a hydrazine group (NH-NH2), and the hydrazine group can also be understood as an amino group.

[0093] In some embodiments, the above step S40 can first resuspend and emulsify the nanoparticles, coenzyme factors, and biological enzymes in an aqueous solution, then add a monomer solution containing an aldehyde group, and perform an immobilization reaction under the catalysis of scandium triflate, and then centrifuge and purify to obtain a covalent organic framework hybrid organelle.

[0094] In some embodiments, the above step S40 does not have any limitation on the specific implementation method for purification, for example, the reaction product after the immobilization reaction can be washed, centrifuged, and dried multiple times. Deionized water, ethanol, methanol, and the like can be used to wash the reaction product, and drying can be performed at high temperature or in a frozen environment.

[0095] In some embodiments, the nanoparticles are preferably one of gold nanoparticles, iron nanoparticles, and copper nanoparticles.

[0096] In some embodiments, the biological enzyme is preferably one of transaminase, glucose dehydrogenase, glutamate dehydrogenase, leucine dehydrogenase, glucose oxidase, catalase, and lipase.

[0097] In some embodiments, the molar ratio of the monomer containing an aldehyde group to the monomer containing a hydrazine group is preferably 2:3.

[0098] In some embodiments, the temperature of the immobilization reaction is preferably 10-40℃; further, the temperature of the immobilization reaction is more preferably 20-30℃.

[0099] In some embodiments, the amount of the biological enzyme added is preferably 0.25-1.0 mg; further, the amount of the biological enzyme added is more preferably 0.5 mg.

[0100] In some embodiments, the amount of scandium triflate added is preferably 10-70 μL.

[0101] In some embodiments, the amount of coenzyme factor added is preferably 5.0 mg.

[0102] The hybrid organelle of the covalent organic framework immobilized with multiple biological and non-biological elements prepared by the present application can effectively improve the tolerance of biological elements to adverse environments, realize protection, integrate the catalytic functions of multiple elements, and realize iterative upgrading of catalytic functions.

[0103] The technical method of the present application will be further described below in combination with specific embodiments.

[0104] Embodiment 1

[0105] This embodiment provides a synthesis method of covalent organic framework material COFcap-2 under different monomer ratios, and the specific steps are as follows:

[0106] A total of 0.03 mmol of 2,5-diethoxybenzene-1,4-di(hydrazinecarboxylate) (DTH-400) containing polyethylene glycol in different proportions (0:30, 1:29, 3:27, 5:25, 10:20, 20:10, 30:0) was added to a reaction vessel containing water (1 mL) and chloroform (7.5 mL), and stirred vigorously at room temperature for 10 min (speed >600 rpm) to obtain reaction solution 1.

[0107] Trimesic aldehyde (TB) (3.3 mg, 0.02 mmol) was dissolved in a mixture of 1.0 mL of chloroform and 1.0 mL of acetonitrile solution to obtain reaction solution 2, which was added to the above reaction solution 1, and then 70 μL of scandium triflate aqueous solution (1.0 g / mL) was added, and stirred vigorously at room temperature for 50 min. After the reaction was completed, the solid was recovered by centrifugation to obtain the covalent organic framework material COFcap-2.

[0108] In order to facilitate understanding of the morphology characteristics of the covalent organic framework material prepared in Example 1, the covalent organic framework material prepared in Example 1 is tested and characterized in the embodiments of the present application. The morphology and crystallinity of COFcap-2 are observed by transmission electron microscopy (TEM), gas adsorption instrument, and powder X-ray diffraction (PXRD). Figure 1 is a schematic diagram of the structure of the covalent organic framework material COF provided by the present application. Figure 2 is a transmission electron microscopy image (a), a powder X-ray diffraction image (b), a gas adsorption image (c), and a schematic diagram of the shell thickness (d) of COFcap-2 formed by different monomer ratios.

[0109] According to Figure 2 It can be seen that COFcap-2 with different monomer ratios, wherein DTH-400:DTH (0:30, 1:29, 3:27, 5:25, 10:20), all have good crystallinity, because the PEG chains can be properly cross-linked in the COF framework without forming PEG chain entanglements, but the PXRD spectrum of DTH-400:DTH (20:10, 30:0) has amorphous, and the BET surface area is low. Moreover, the introduction of DTH-400 promotes the formation of hollow COF capsules, and the increase in the proportion of DTH-400 causes the capsule wall to gradually thin.

[0110] Example 2

[0111] The present embodiment provides a synthesis method of COFcap-2 under different solvent ratios, and the specific implementation steps are as follows:

[0112] DTH-400 (0.003 mmol), DTH (0.027 mmol) were added to different solvent systems, wherein the ratio of water: chloroform was 0.5:7.5, 1.0:7.5, 2.0:7.5, 3.0:7.5, and the solution was stirred vigorously at room temperature for 10 min (rotation speed >600 rpm) in a 50 mL bottle to obtain solution A. TB (3.3 mg, 0.02 mmol) was dissolved in a mixed solution of 1.0 mL chloroform and 1.0 mL acetonitrile, and the solution was added to the above solution A, followed by adding 70 μL of a scandium triflate aqueous solution (concentration of 1.0 g / mL), and stirring vigorously at room temperature for 50 min. After the reaction was completed, the solid was recovered by centrifugation to obtain the covalent organic framework material COFcap-2.

[0113] In order to facilitate understanding of the morphology characteristics of the covalent organic framework material prepared in Example 2, the covalent organic framework material prepared in Example 2 is tested and characterized in the embodiments of the present application. The morphology and crystallinity of COFcap-2 are observed by transmission electron microscopy (TEM), gas adsorption instrument, and powder X-ray diffraction (PXRD). Figure 3are TEM images (a) and dynamic light scattering images (b) of COFcap-2 formed by different solvent ratios. The morphology and size of COFcap-2 were observed by transmission electron microscopy (TEM) and dynamic light scattering (DLS).

[0114] According to Figure 3 It can be seen that the size of the COFcap-2 capsule gradually increases with the increase of the proportion of water.

[0115] Example 3

[0116] This example provides a method for immobilizing nanoparticles by COFcap-2, and the specific steps are as follows:

[0117] DTH-400 (0.003 mmol), DTH (0.027 mmol), and a nanoparticle dispersion solution (300 uL) were added to a 50 mL bottle containing water (1 mL) and chloroform (7.5 mL), and stirred vigorously (rotation speed > 600 rpm) at room temperature for 10 min. TB (3.3 mg, 0.02 mmol) was dissolved in a solution of 1.0 mL chloroform and 1.0 mL acetonitrile, and the solution was added to the above solution, followed by the addition of 70 uL of a scandium triflate aqueous solution (concentration of 1.0 g / mL). The reaction was stirred vigorously at room temperature for 50 min, and after the reaction was completed, the solid was recovered by centrifugation to obtain the immobilized product.

[0118] In order to facilitate the understanding of the morphology characteristics of the immobilized product prepared in Example 3, the immobilized product prepared in Example 3 was tested and characterized in this example. Figure 4 is a transmission electron microscope image of the product prepared in Example 3. The morphology of COFcap-2 was observed by transmission electron microscopy (TEM).

[0119] According to Figure 4 It can be seen that COFcap-2 can encapsulate nanoparticles inside the material.

[0120] Example 4

[0121] This example provides a method for immobilizing coenzyme factors (NAD + ) by COFcap-2, and the specific steps are as follows:

[0122] DTH-400 (0.003 mmol), DTH (0.027 mmol), and NAD +A 50 mL vial containing DTH-400 (5.0 mg) was added with water (1 mL) and chloroform (7.5 mL) and stirred vigorously at room temperature for 10 min (rotation speed > 600 rpm). TB (3.3 mg, 0.02 mmol) was dissolved in 1.0 mL chloroform and 0.1 mL acetonitrile solution, which was added to the above solution, followed by the addition of 70 μΐ^of scandium triflate aqueous solution (1.0 g / mL), and stirred vigorously at room temperature for 50 min. After the completion of the reaction, the solid was recovered by centrifugation to obtain the immobilized product.

[0123] In order to facilitate the understanding of the morphology of the immobilized product prepared in Example 4, the immobilized product prepared in Example 4 was tested and characterized in this example. X-ray photoelectron spectroscopy (XPS) was used to characterize the NAD + The leakage of NAD + was also tested. Figure 5 is the X-ray photoelectron spectrogram of the product prepared in Example 4 (a) and the NAD + leakage image (b).

[0124] According to Figure 5 , COFcap-2 successfully encapsulates NAD + and has a lower leakage rate (less than 1.3%).

[0125] Example 5

[0126] This example provides a method for the immobilization of lipase by COFcap-2 with different monomer ratios, and the specific steps are as follows:

[0127] A 50 mL vial containing DTH-400: DTH (0:30, 1:29, 3:27, 5:25, 10:20) in different ratios with a total molar amount of 0.03 mmol and 6 mg of lipase was added with water (1 mL) and chloroform (7.5 mL) and stirred vigorously at room temperature for 10 min (rotation speed > 600 rpm). TB (3.3 mg, 0.02 mmol) was dissolved in 1.0 mL chloroform and 0.1 mL acetonitrile solution, which was added to the above solution, followed by the addition of 10 μΐ^of scandium triflate aqueous solution (1.0 g / mL), and stirred vigorously at room temperature for 50 min. After the completion of the reaction, the product was collected by centrifugation at a rotation speed of 5500 rpm, washed with deionized water for 3 times, and dried at room temperature to obtain Lipase@COFcap-2 formed by different monomer ratios.

[0128] After the encapsulation and washing process, the supernatant of each operation was collected, and the concentration of protein was quantified according to the standard protocol of Bradford determination, and the residual enzyme activity of free enzyme and immobilized Lipase@COFcap-2 was determined. Figure 6Enzyme loading (a) and enzyme activity (b) of Lipase® COFcap-2 formed by different monomer ratios.

[0129] According to Figure 6 It can be seen that when DTH-400: DTH is 3:27, the optimal enzyme activity, enzyme loading and enzyme loading rate are obtained.

[0130] Example 6

[0131] This example provides a method for immobilizing glutamate dehydrogenase (GluDH) on COFcap-2, and the specific steps are as follows:

[0132] DTH-400 (0.003 mmol), DTH (0.027 mmol), and GluDH (0.25, 0.50, or 1.00 mg) were added to a 50 mL bottle containing water (1 mL) and chloroform (7.5 mL), and stirred vigorously at room temperature for 10 min (rotation speed > 600 rpm). TB (3.3 mg, 0.02 mmol) was dissolved in 1.0 mL of chloroform and 0.1 mL of acetonitrile solution, which was added to the above solution, followed by the addition of 10 μL of scandium triflate aqueous solution (1.0 g / mL), and stirred vigorously at room temperature for 50 min. After the reaction was completed, the product was collected by centrifugation at a rotation speed of 5500 rpm, washed with deionized water three times, and dried at room temperature to obtain GluDH® COFcap-2 formed by different amounts of GluDH addition.

[0133] After the encapsulation and washing processes, the supernatant of each operation was collected, and the concentration of protein was quantified according to the standard protocol of Bradford determination, while the residual enzyme activity of free enzyme and immobilized GluDH® COFcap-2 was determined. Figure 7 Enzyme loading (a) and enzyme activity (b) of GluDH® COFcap-2.

[0134] According to Figure 7 It can be seen that when the amount of GluDH added is 0.50 mg, the optimal enzyme activity, enzyme loading and enzyme loading rate are obtained.

[0135] Example 7

[0136] This example provides a method for immobilizing glucose dehydrogenase (GCDH) on COFcap-2, and the specific steps are as follows:

[0137] A 50 mL vial containing DTH-400 (0.003 mmol), DTH (0.027 mmol), GCDH (0.25 mg, 0.50 mg, 1.00 mg) was added with water (1 mL) and chloroform (7.5 mL), and stirred vigorously (speed > 600 rpm) at room temperature for 10 min. TB (3.3 mg, 0.02 mmol) was dissolved in 1.0 mL chloroform and 0.1 mL acetonitrile, and the solution was added to the above solution, followed by the addition of 10 μL scandium triflate aqueous solution (1.0 g / mL), and stirred vigorously at room temperature for 50 min. After the completion of the reaction, the product was collected by centrifugation at a speed of 5500 rpm, washed with deionized water for 3 times, and dried at room temperature to obtain GCDH◎COFcap-2 formed by different amounts of GCDH.

[0138] After the encapsulation and washing process, the supernatant of each operation was collected, and the concentration of protein was quantified according to the standard protocol of Bradford determination, while the residual enzyme activity of free enzyme and immobilized GCDH◎COFcap-2 was determined. Figure 8 The enzyme loading (a) and enzyme activity (b) of GCDH◎COFcap-2.

[0139] According to Figure 8 It can be seen that when the amount of GCDH added is 0.50 mg, the enzyme activity, enzyme loading and enzyme loading rate are optimal.

[0140] Example 8

[0141] This example provides a method for immobilizing COFcap-2 on transaminase (ω-TA), and the specific steps are as follows:

[0142] A 50 mL vial containing DTH-400 (0.003 mmol), DTH (0.027 mmol), ω-TA (0.25 mg, 0.50 mg, 1.00 mg) was added with water (1 mL) and chloroform (7.5 mL), and stirred vigorously at room temperature for 10 min (speed > 600 rpm). TB (3.3 mg, 0.02 mmol) was dissolved in 1.0 mL chloroform and 0.1 mL acetonitrile, and the solution was added to the above solution, followed by the addition of 10 μL scandium triflate aqueous solution (1.0 g / mL), and stirred vigorously at room temperature for 50 min. After the completion of the reaction, the product was collected by centrifugation at a speed of 5500 rpm, washed with deionized water for 3 times, and dried at room temperature to obtain ω-TA◎COFcap-2 formed by different amounts of ω-TA.

[0143] After the encapsulation and washing process, the supernatant of each operation was collected and the concentration of protein was quantified according to the standard protocol of Bradford determination, while the residual enzyme activity of free enzyme and immobilized ω-TA COFcap-2 was determined. Figure 9 is the enzyme loading (a) and enzyme activity (b) of ω-TA COFcap-2.

[0144] According to Figure 9 It can be seen that when the amount of ω-TA added is 0.50 mg, the enzyme activity, enzyme loading and enzyme loading rate are optimal.

[0145] Example 9

[0146] The present application provides a method for co-immobilization of COFcap-2 on GluDH, GCDH and ω-TA, and the specific implementation steps are as follows:

[0147] FITC-fluorescein-labeled GCDH to obtain FITC-GCDH, RhB-fluorescein-labeled GluDH to obtain RhB-GluDH, and DAPI-fluorescein-labeled ω-TA to obtain DAPI-ω-TA. DTH-400 (0.003 mmol), DTH (0.027 mmol), FITC-GCDH (0.30 mg), RhB-GluDH (0.30 mg), and DAPI-ω-TA (0.25 mg) were added to a 50 mL bottle containing water (1 mL) and chloroform (7.5 mL), and stirred vigorously (speed > 600 rpm) at room temperature for 10 min. TB (3.3 mg, 0.02 mmol) was dissolved in 1.0 mL of chloroform and 0.1 mL of acetonitrile solution, which was added to the above solution, followed by the addition of 10 μL of scandium triflate aqueous solution (1.0 g / mL), and the solution was stirred vigorously at room temperature for 50 min. After the reaction was completed, the solid was recovered by centrifugation to obtain a co-immobilized multi-enzyme biocatalyst.

[0148] The co-immobilization of COFcap-2 on FITC-GCDH, RhB-GluDH and DAPI-ω-TA was observed by confocal laser scanning microscope (CLSM). Figure 10 is the confocal laser scanning microscope image of the co-immobilization of COFcap-2 on GluDH, GCDH and ω-TA.

[0149] According to Figure 10 It can be seen that COFcap-2 uniformly immobilizes FITC-GCDH, RhB-GluDH and DAPI-ω-TA inside the COF cavity to prepare a co-immobilized multi-enzyme biocatalyst.

[0150] Example 10

[0151] This example provides a test of the enzyme activity of Lipase and Lipase COFcap-2. The specific implementation steps are as follows:

[0152] For free lipase, 50 μL of lipase solution (6.0 mg / mL) and 100 μL of nitrophenyl acetate acetonitrile solution (p-NPA, 100 mM) were added to PB buffer (50 mM, pH = 7.0) diluted to a final volume of 3.0 mL. Then, the reaction kinetics of the free lipase was measured at 402 nm in a thermostatic stirring ultraviolet-visible spectrophotometer. The Lipase COFcap-2 material containing 0.3 mg of lipase was mixed with 100 μL of p-NPA solution, and then diluted to 3.0 mL with PB buffer to determine the activity of Lipase COFcap-2. Then the same method as the free enzyme was used to determine the activity by ultraviolet-visible light.

[0153] The above test results show that the immobilized Lipase COFcap-2 material can maintain > 84% of the enzyme activity.

[0154] Example 11

[0155] This example provides a test of the enzyme activity of GCDH and GCDH COFcap-2. The specific implementation steps are as follows: free GCDH and GCDH COFcap-2 containing the same mass of GCDH were added to a mixed solution of 3 mL of PB buffer (50 mM, pH = 7.0), 0.16 mM NADH, and 5 mM glucose. In an ultraviolet-visible spectrophotometer with constant temperature stirring, the increase in absorbance value at 340 nm was tested to determine the enzyme activity. + and 5mM glucose. In an ultraviolet-visible spectrophotometer with constant temperature stirring, the increase in absorbance value at 340 nm was tested to determine the enzyme activity.

[0156] The test results of Example 11 show that the immobilized GCDH COFcap-2 material can maintain > 77% of the enzyme activity

[0157] Example 12: Test of the enzyme activity of GluDH and GluDH COFcap-2. The specific implementation steps are as follows: free GluDH and GluDH COFcap-2 containing the same amount of GluDH were added to a mixed solution of 3 mL of PB buffer (50 mM, pH = 7.0), 0.16 mM NADH, 5 mM 2-ketoglutaric acid, and 100 mM NH4Cl. In an ultraviolet-visible spectrophotometer with constant temperature stirring, the decrease in absorbance value at 340 nm was tested to determine the enzyme activity.

[0158] The test results of the above Example 12 show that the immobilized GluDH@COFcap-2 material can maintain >74% of the enzyme activity

[0159] Example 13

[0160] This example provides an enzyme activity test of ω-TA and ω-TA@COFcap-2, and the specific implementation steps are as follows:

[0161] The same amount of free ω-TA or ω-TA@COFcap-2 containing ω-TA was added to 9.8 mL PB buffer (50 mM, pH = 7.0) containing glutamic acid (20 mM) and coenzyme pyridoxal 5'-phosphate monohydrate (PLP, 1.0 mM). Then, 200 μL of 4-phenyl-2-butanone solution (20 mg / mL DMSO solution) was added to the above solution, and the resulting mixture was shaken at 30°C for 24 h. Then, the solution was extracted with ethyl acetate (2 x 500 μL). The organic layer was separated by centrifugation (3 min, 8000 rpm), and finally Na2SO4 was added for drying. After conventional derivatization of the sample using acetic anhydride (2 μL / mg substrate), di-tert-butyl dicarbonate (50 μL ethyl acetate solution / mg substrate) or benzyl chloroformate (2 μL / mg substrate), the enantiomeric excess of the corresponding amine was determined by gas chromatography-mass spectrometer (GC-MS) or high performance liquid chromatography (HPLC).

[0162] The test results of the above Example 13 show that the immobilized ω-TA@COFcap-2 material can maintain >78% of the enzyme activity

[0163] Example 14

[0164] This example provides an ammonia production performance test of Au@COFcap-2, and the specific implementation steps are as follows:

[0165] Electrochemical measurements were performed on an electrochemical station using a three-electrode system. Au@COFcap-2, Fe2O3@COFcap-2 and Cu@COFcap-2 were used as the working electrode, Ag / AgCl (saturated KCl) and Pt plate as the reference electrode and counter electrode, respectively. The electrolyte was 0.1 M Na2SO4 aqueous solution. Before each NRR process, the electrochemical device was first continuously bubbled with N2 gas for at least 1 h. Then, the gas was continuously introduced during the entire electrochemical reaction process, and the potentiometric measurement was carried out at a series of applied potentials, including -0.2, -0.3, -0.4, -0.5 and -0.6 V vs Ag / AgCl, at room temperature for 2 h. Figure 11 is the ammonia production performance of Au@COFcap-2.

[0166] According toFigure 11 Au◎COFcap-2 has the highest ammonia yield of 6.5 mM h at -0.4 V vs Ag / AgCl. –1 g –1 Au◎COFcap-2 has the highest faradaic efficiency of 45% at -0.3 V vs Ag / AgCl.

[0167] Example 15

[0168] This example provides a method for co-immobilizing multiple chemical and biological catalysts to synthesize catalyst◎COFcap-2, the specific implementation steps are as follows:

[0169] DTH-400 (0.003 mmol), DTH (0.027 mmol), nanoparticle dispersion (300 uL), NAD + (5.0 mg), glutamate dehydrogenase (0.30 mg), glucose dehydrogenase (0.30 mg), transaminase (0.25 mg) were added to a 50 mL bottle containing water (1 mL) and chloroform (7.5 mL), and stirred vigorously at room temperature for 10 min (rotation speed > 600 rpm). TB (3.3 mg, 0.02 mmol) was dissolved in 1.0 mL of chloroform and 0.1 mL of acetonitrile solution, which was added to the above solution, followed by adding 10 uL of scandium triflate aqueous solution (1.0 g / mL), and stirring vigorously at room temperature for 50 min. After the reaction was completed, the solid was recovered by centrifugation, and a 0.2 wt% Nafion solution was added to make a catalyst◎COFcap-2 paste, which was coated on the surface of carbon paper to prepare a working electrode for catalytic conversion of nitrogen to chiral amines. Figure 12 The performance of catalyst◎COFcap-2 in catalytic conversion of nitrogen to chiral amines.

[0170] According to Figure 12 It can be seen that the synthesized catalyst◎COFcap-2 has good catalytic conversion ability.

[0171] Example 16

[0172] The effect of different concentrations of biological catalysts on the cascade catalytic activity of catalyst◎COFcap-2, the specific implementation steps are as follows:

[0173] DTH-400 (0.003 mmol), DTH (0.027 mmol), nanoparticle dispersion (300 uL), NAD +(5.0 mg), GluDH, GCDH, ω-TA (a, b, c groups represent the added amount of GluDH + GCD + ω-TA, a = 0.15 + 0.15 + 0.125 mg; b = 0.30 + 0.30 + 0.25 mg; c = 0.6 + 0.6 + 0.5 mg) were added to a 50 mL bottle containing water (1 mL) and chloroform (7.5 mL) and stirred vigorously at room temperature for 10 min (rotation speed > 600 rpm). TB (3.3 mg, 0.02 mmol) was dissolved in 1.0 mL of chloroform and 0.1 mL of acetonitrile solution, which was added to the above solution, followed by the addition of 10 μL of scandium triflate aqueous solution (1.0 g / mL), and stirred vigorously at room temperature for 50 min. After the reaction was completed, the solid was recovered by centrifugation, and a 0.2 wt% Nafion solution was added to prepare a catalyst◎COFcap-2 paste to coat the surface of carbon paper to prepare a working electrode for catalytic conversion of nitrogen to chiral amines. Figure 13 The performance of catalyst◎COFcap-2 formed by different ratios of biological catalysts for catalytic conversion of nitrogen to chiral amines.

[0174] According to Figure 13 It can be seen that the synthesized catalyst◎COFcap-2 of group b has better catalytic conversion capacity than catalyst◎COFcap-2 of group a and catalyst◎COFcap-2 of group c, because higher enzyme concentration can cause repeated collisions and reduce overall performance.

[0175] Example 17

[0176] The catalytic performance of catalyst◎COFcap-2 for different catalytic substrates was tested, and the specific implementation steps were as follows:

[0177] The in vitro catalytic system synthesis of chiral amine drug molecules was carried out in a H-shaped two-chamber bioelectrochemical reactor at 30℃ under a constant potential of -0.4V vs Ag / AgCl. A paste of catalyst◎COFcap-2 containing 0.2wt% Nafion was coated on carbon paper as the working electrode before air-drying. The electrolyte was an aqueous 0.1M Na2SO4 solution containing 1mM PLP, 5mM glucose, 1mM 2-ketoglutarate, 1mM ketone substrate and 1% DMSO. The potentiostatic measurement was carried out at room temperature under -0.4V vs Ag / AgCl for 24h. The reaction mixture was extracted with ethyl acetate. The organic layer was separated by centrifugation and finally dried with Na2SO4. After conventional derivatization of the sample using acetic anhydride (2μL / mg substrate), di-tert-butyl dicarbonate (50μL ethyl acetate solution / mg substrate) or benzyl chloroformate (2μL / mg substrate), the enantiomeric excess of the corresponding amine was determined by gas chromatography-mass spectrometer (GC-MS) or high performance liquid chromatography (HPLC). Figure 14 is the performance of catalyst◎COFcap-2 in catalytic conversion of nitrogen to different chiral amines.

[0178] According to Figure 14 It can be seen that the synthesized catalyst◎COFcap-2 has good catalytic conversion ability for a variety of substrates.

[0179] Example 18

[0180] The recycling performance of catalyst◎COFcap-2 is as follows:

[0181] The carbon paper coated with catalyst◎COFcap-2 was used as the working electrode, after the reaction, the working electrode was washed twice with phosphate buffer, and then the reaction was carried out again according to the reaction system of Example 17. After the reaction, the conversion rate and residual activity of each batch were determined, and the ratio of residual enzyme activity of the fifteenth reaction to that of the first reaction was the residual enzyme activity after fifteen times of reuse. In this way, the catalytic efficiency of catalyst◎COFcap-2 co-immobilized catalyst and the change of residual activity with the increase of the number of uses were investigated. Figure 15 is the recycling performance of catalyst◎COFcap-2.

[0182] The results are shown in Figure 15 The relative yield of catalyst◎COFcap-2 co-immobilized catalyst is still higher than 60% after fifteen times of reuse. The experimental difficulty of the whole catalytic system is low, the overall conversion efficiency is high, and the cost can be further reduced.

[0183] The present application introduces a PEG-driven emulsion template method to prepare COFcap-2, which can one-pot encapsulate enzymes (e.g., ω-transaminase, glutamate dehydrogenase, and glucose dehydrogenase), chemical catalysts (i.e., metal nanoparticles), and coenzymes (NAD+), forming a co-immobilized enzyme-chemical catalyst, providing an innovative, customizable platform for multi-enzyme catalysis and biomanufacturing, thus expanding the possibilities for high-performance applications in various industries. In addition, this strategy not only opens up a new way for green and sustainable synthesis of pharmaceutical molecules, but also provides an energy-saving alternative for biotechnological applications.

[0184] The present application COFcap-2 combines chemical catalysis (NRR) and biological catalysis (enzyme cascade) to achieve N2 fixation into homochiral amines, such as the drug compound (R)-sitagliptin, which is the active ingredient in Januvia®

[0185] The 11 substrates in the examples of the present application verify the performance of COFcap-2, each substrate has good catalytic performance and excellent enantioselectivity, such as 2-amino-4-phenylbutane (62 μM·h -1 The yield, >99% ee). The performance of COFcap-2 is superior to that of the reported homogeneous catalysts, and maintains good relative yield after 15 cycles.

[0186] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical scope disclosed in the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.​

Claims

1. A method of preparing a covalent organic framework material, characterized in that, The method comprises the following steps: dissolving monomers containing hydrazide functional groups and 2,5-bis(2-methoxyethoxy)terephthalic dihydrazide containing polyethylene glycol in an organic solvent aqueous solution to form a dispersion; adding a monomer solution containing aldehyde functional groups to the dispersion, and performing a fixation reaction under the condition of a catalyst, and then centrifuging and purifying to obtain a covalent organic framework material; the monomer containing hydrazide functional groups is at least one of 2,5-diethoxybenzene-1,4-bis(formylhydrazide), 2,5-bis(2-methoxyethoxy)terephthalic dihydrazide, 2,5-bis(but-3-en-1-oxy)terephthalic dihydrazide, and 2-(but-3-en-1-oxy)-5-(2-methoxyethoxy)terephthalic dihydrazide; the monomer containing aldehyde functional groups is at least one of trimesic aldehyde, 2,6-dihydroxy-1,3,5-benzene tricarboxaldehyde, and tricarboxaldehyde m-phenol; the catalyst is at least one of a compound containing anhydride functional groups, a compound containing carboxylic acid functional groups, a compound containing imidazole functional groups, a compound containing hydroxyl functional groups, and scandium triflate.

2. The method of claim 1, wherein, the molecular weight of polyethylene glycol in the 2,5-bis(2-methoxyethoxy)terephthalic dihydrazide containing polyethylene glycol is at least one of 400 g / mol, 600 g / mol, and 800 g / mol.

3. The method of claim 1, wherein, the molar ratio of the monomer containing hydrazide functional groups, the 2,5-bis(2-methoxyethoxy)terephthalic dihydrazide containing polyethylene glycol, and the monomer containing aldehyde functional groups is 0:30:20, 1: 29：20、3：27：20、5：25：20、10：20：20、20：10：20、30：0：20； and / or, the volume ratio of water and organic solvent in the organic solvent aqueous solution is 0.5:7.5, 1.0:7.5, 2.0:7.5, or 3.0:7.

5.

4. The method of claim 3, wherein, the molar ratio of the monomer containing hydrazide functional groups, the 2,5-bis(2-methoxyethoxy)terephthalic dihydrazide containing polyethylene glycol, and the monomer containing aldehyde functional groups is 3:27:20; and / or, the volume ratio of water and organic solvent in the organic solvent aqueous solution is 1.0:7.5; and / or, the catalyst is at least one of acetic acid and scandium triflate.

5. A covalent organic framework material prepared by the method according to any one of claims 1-4, for use in the preparation of a covalent organic framework hybrid organelle.

6. A method of producing the covalent organic framework hybrid organelle of claim 5, wherein, The method comprises the following steps: dissolving monomers containing aldehyde functional groups and monomers containing hydrazide functional groups in an organic solvent aqueous solution to form a dispersion; after resuspending and emulsifying nanoparticles, coenzyme factors, and biological enzymes in an aqueous solution, adding a monomer solution containing aldehyde functional groups, and performing a fixation reaction under the condition of scandium triflate as a catalyst, and then centrifuging and purifying to obtain a covalent organic framework hybrid organelle.

7. The method of claim 6, wherein, the monomer containing aldehyde functional groups is at least one of trimesic aldehyde, 2,6-dihydroxy-1,3,5-benzene tricarboxaldehyde, and tricarboxaldehyde m-phenol; and / or the monomer containing a hydrazide functional group is at least one of 2,5-bis(2-methoxyethoxy)terephthalic hydrazide, 2,5-bis(but-3-en-1-oxy)terephthalic hydrazide, 2-(but-3-en-1-oxy)-5-(2-methoxyethoxy)terephthalic hydrazide; and / or the organic solvent is chloroform; and / or the nanoparticle is at least one of gold nanoparticle, iron nanoparticle, copper nanoparticle; and / or the biological enzyme is at least one of transaminase, glucose dehydrogenase, glutamate dehydrogenase, leucine dehydrogenase, glucose oxidase, catalase, lipase.

8. The method of claim 7, wherein, The molar ratio of the monomer containing an aldehyde functional group to the monomer containing a hydrazide functional group is 2:3; and / or the temperature of the immobilization reaction is 10-40℃; and / or the amount of the biological enzyme added is 0.25-1.0 mg; and / or the amount of scandium triflate added is 10-70 μL; and / or the amount of coenzyme factor added is 5.0 mg.

9. The method of claim 8, wherein, The amount of the biological enzyme added is 0.5 mg; and / or the temperature of the immobilization reaction is 20-30℃.

10. Use of the covalent organic framework hybrid organelle prepared by the method of any one of claims 6-9 for producing a pharmaceutically active ingredient or a pharmaceutical intermediate.

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