Covalent organic framework material, preparation method thereof, hybrid organelle prepared from covalent organic framework material, preparation method and application

Abstract

The invention discloses a covalent organic framework material, a preparation method of the covalent organic framework material, a hybrid organelle prepared from the covalent organic framework material, a preparation method and application, and relates to the field of bio-enzyme engineering. The preparation method comprises the following steps: dissolving 2, 5-bis (2-methoxyethoxy) terephthaloyl hydrazine in an aqueous solution of an organic solvent to form a dispersion liquid; and adding a monomer solution containing an aldehyde functional group into the dispersion liquid, carrying out an immobilization reaction in the presence of a catalyst, and carrying out centrifugal purification to obtain the covalent organic framework material. Therefore, the covalent organic framework material COFcap-2 is prepared by introducing a PEG-driven emulsion template method, and the covalent organic framework material can fix various biological elements, so that the tolerance of the biological elements to a bad environment is improved, and a protection effect is realized.

Description

Technical Field

[0001] The present application relates to the field of bioenzyme engineering technology, and in particular to a covalent organic framework material and a preparation method thereof, a hybrid organelle prepared from the covalent organic framework material, a preparation method and an application thereof. Background Art

[0002] Using CO2 and N2 as raw materials and clean energy such as electricity / light energy to synthesize materials is an important path to save fossil resources, protect the environment, and achieve green manufacturing, and it is also the focus of international scientific and technological competition. Most of the existing manufacturing routes are concentrated on the reduction of CO2 and N2 to synthesize organic one-carbon / one-nitrogen molecules. However, multi-carbon / multi-nitrogen compounds have wider applications in biomedicine, pesticides, fine chemicals and materials science. Therefore, the manufacturing route of CO2 and N2 reduction to synthesize high-value-added multi-carbon / multi-nitrogen compounds urgently needs to be broken through.

[0003] At present, it is difficult to achieve such complex catalytic processes with a single biological or non-biological element. The production of multi-carbon / multi-nitrogen compounds can be completed through a composite catalytic system that rationally integrates multiple elements. However, homogeneous catalytic systems often face problems such as low mass transfer rate, low conversion efficiency, poor stability, and difficulty in recycling. Summary of the invention

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

[0005] In order to achieve the above purpose, the technical solution of the embodiment of the present application is:

[0006] The first aspect of the present application provides a method for preparing a covalent organic framework material, comprising:

[0007] Dissolving a monomer containing a hydrazide functional group and 2,5-bis(2-methoxyethoxy)terephthaloylhydrazide containing polyethylene glycol in an organic solvent aqueous solution to form a dispersion;

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

[0009] In combination with the first aspect, preferably, the monomer containing a hydrazide functional group is at least one of 2,5-diethoxybenzene-1,4-di(formyl hydrazide), 2,5-bis(2-methoxyethoxy)terephthaloyl hydrazide, 2,5-bis(but-3-ene-1-oxy)terephthaloyl hydrazide and 2-(but-3-ene-1-oxy)-5-(2-methoxyethoxy)terephthaloyl hydrazide;

[0010] And / or, the molecular weight of the polyethylene glycol in the 2,5-bis(2-methoxyethoxy)terephthaloyl hydrazide containing polyethylene glycol is at least one of 400 g / mol, 600 g / mol, and 800 g / mol;

[0011] And / or, the monomer containing aldehyde functional group is at least one of trimesaldehyde, 2,6-dihydroxy-1,3,5-benzenetricarbaldehyde and trialdehyde phloroglucinol;

[0012] And / or, 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, and a compound containing hydroxyl functional groups.

[0013] In combination with the first aspect, preferably, the molar ratio of the monomer containing a hydrazide functional group, the 2,5-bis(2-methoxyethoxy)terephthaloyl hydrazide containing polyethylene glycol, and the monomer containing an aldehyde 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] And / or, the volume ratio of water to organic solvent in the organic solvent aqueous solution is 0.5:7.5, 1.0:7.5, 2.0:7.5, 3.0:7.5.

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

[0016] and / or, the volume ratio of water to organic solvent in the organic solvent aqueous solution is 1.0:7.5;

[0017] And / or, the catalyst is at least one of acetic acid and scandium trifluoromethanesulfonate.

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

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

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

[0021] After the nanoparticles, coenzyme factors and biological enzymes are resuspended and emulsified in an aqueous solution, a monomer solution containing an aldehyde functional group is added, and an immobilization reaction is carried out under the condition that scandium trifluoromethanesulfonate is used as a catalyst. After centrifugation and purification, a covalent organic framework hybrid organelle is obtained.

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

[0023] and / or at least one of the 2,5-bis(2-methoxyethoxy)terephthaloyl hydrazide, 2,5-bis(but-3-ene-1-oxy)terephthaloyl hydrazide and 2-(but-3-ene-1-oxy)-5-(2-methoxyethoxy)terephthaloyl 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 hydrazide functional group is 2:3;

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

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

[0030] And / or, the amount of scandium trifluoromethanesulfonate added is 10 to 70 μL;

[0031] And / or, the added amount of the coenzyme factor is 5.0 mg.

[0032] In combination with the third aspect, preferably, the added 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 an application of a covalent organic framework hybrid organelle prepared by the method described in the third aspect in the production of active pharmaceutical ingredients or pharmaceutical intermediates.

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

[0036] The present application introduces a PEG-driven emulsion template method to prepare a covalent organic framework material C OFcap-2, which can fix a variety of biological elements, improve their tolerance to adverse environments, and achieve a protective effect.

[0037] The present application can utilize the covalent organic framework material COFcap-2 to co-immobilize various enzymes, nanoparticles and coenzyme factors, and the prepared covalent organic framework hybrid organelle electroenzymatic catalysis generates various chiral amines, with higher catalytic efficiency, more excellent stability and recyclability than pure enzyme catalytic system (nitrogenase, transaminase, diaphorase, L-alanine dehydrogenase). The covalent organic framework co-immobilizes the hybrid organelles of biological and non-biological elements, and since various elements are co-fixed inside the hybrid organelles, the spatial distance can be shortened, and the cascade catalytic reaction can be effectively promoted, and energy efficient utilization is achieved, as well as efficient conversion from cheap energy to high value-added products is achieved. In addition, the tolerance of enzymes to adverse environments is effectively improved, and a protective effect is achieved, while having excellent recyclability.

[0038] The present application uses a self-assembly strategy to co-immobilize a variety of enzymes, nanoparticles and coenzyme factors. A variety of enzymes and a variety of chemical catalysts are mixed with a monomer 1 containing a hydrazide functional group and a monomer containing an aldehyde functional group in a reaction solvent, and a condensation reaction is carried out under the condition of scandium trifluoromethanesulfonate as a catalyst, and centrifugation and washing are performed to obtain a co-immobilized enzyme-chemical catalyst. The hybrid organelles of co-immobilized biological-non-biological elements include but are not limited to applications in the production of active pharmaceutical ingredients or pharmaceutical intermediates and other medical fields, and have excellent catalytic activity, stability and recyclability. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0040] Figure 2 Transmission electron microscopy images (a), powder X-ray diffraction images (b), gas adsorption images (c) and shell thickness schematic diagram (d) of COFcap-2 formed with different monomer ratios;

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

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

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

[0044] Figure 6 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] Fig. 9 is the enzyme loading (a) and enzyme activity (b) of ω-TA◎COFcap-2;

[0048] Fig.10 This is a laser confocal microscopy image of COFcap-2 co-immobilizing GluDH, GCDH and ω-TA;

[0049] Fig.11 is the ammonia production performance of Au◎COFcap-2;

[0050] Fig.12 The performance of catalyst◎COFcap-2 in catalyzing the conversion of nitrogen into chiral amines;

[0051] Fig.13 The performance of catalyst◎COFcap-2 formed by different biocatalyst ratios in catalyzing the conversion of nitrogen into chiral amines;

[0052] Fig.14 The performance of catalyst◎COFcap-2 in catalyzing the conversion of nitrogen into different chiral amines;

[0053] Fig.15 It is the recyclability of catalyst◎COFcap-2. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in conjunction with the accompanying drawings. The described embodiments should not be regarded as limiting the present application. All other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of this application.

[0055] In the following description, reference is made to "some embodiments", which describe a subset of all possible embodiments, but it is understood that "some embodiments" may be the same subset or different subsets of all possible embodiments, and may be combined with each other without conflict. Unless otherwise defined, all technical and scientific terms used in the embodiments of the present application have the same meaning as those commonly understood by those skilled in the art of the technical field of the embodiments of the present application. The terms used in the embodiments of the present application are only for the purpose of describing the embodiments of the present application and are not intended to limit the present application.

[0056] In the following description of this embodiment, the terms "include", "comprising", "having" and "containing" are all open terms, meaning including but not limited to.

[0057] It should be noted that all raw materials / reagents in the examples of the present application 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 examples of the present application is only used to describe the association relationship between associated objects, indicating that three relationships may exist, for example, A and / or B means the existence of A alone, B alone, and A and B at the same time, among which A and B can be singular or plural, and the character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0058] In the following description of this embodiment, the term "at least one" refers to one or more, and "plurality" refers to two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b or c", or "at least one of a, b and c", can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

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

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

[0061] Those skilled in the art will appreciate that the numerical ranges in the examples of the present application are to be understood as also specifically disclosing each intermediate value between the upper and lower limits of the range. Each smaller range between the intermediate values ​​in any stated value and stated range and any other stated value or intermediate value in the range is also included in the present application. The upper and lower limits of these smaller ranges may be independently included or excluded in the scope.

[0062] Unless otherwise stated, the technical / scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the field described in the present application. Although the present application only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the embodiments or test cases of the present application. All documents mentioned in this specification are generally incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of the present application shall prevail.

[0063] It should be noted that all raw materials and / or reagents in the examples 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-fixing enzymes and chemical catalysts in covalent organic framework (COF) capsules (COFcaps). Combining the properties of enzyme molecules and chemical catalysts themselves, the effective assembly of enzyme molecules and chemical catalysts under mild conditions is explored and studied, and the tolerance to harsh conditions such as organic solvents, proteases, and high temperatures is effectively improved while maintaining the activity of enzyme molecules and chemical catalysts. The resulting bioelectrocatalytic system is heterogeneous, can effectively convert N2 into chiral amines, and is easy to recycle. In addition, the present application not only realizes the immobilization of various enzyme molecules and chemical catalysts by COFs under mild conditions, but also develops a biological-non-biological composite catalytic system with material energy flow synergistic function, while using the idea of ​​process engineering integration to shift from the development of a single technology / strategy to platform construction.

[0065] In a first aspect, the present application provides a method for preparing a covalent organic framework material, the method comprising:

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

[0067] S20, adding a monomer solution containing an aldehyde functional group to the dispersion, performing an immobilization reaction under a catalyst condition, and obtaining a covalent organic framework material after centrifugal purification.

[0068] In the present application, a method for synthesizing a plurality of components using a covalent organic framework material (COFs) is provided, wherein 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, a glucose dehydrogenase, a leucine dehydrogenase, a glutamate dehydrogenase, a glucose oxidase, a catalase, or a lipase, the nanoparticles are gold nanoparticles, iron nanoparticles, or copper nanoparticles, and the coenzyme factor is NAD + and NADH.

[0069] This strategy uses covalent organic framework materials to fix a variety of biological elements (enzymes), improve their tolerance to adverse environments, and achieve a protective effect.

[0070] It should be noted that the monomer containing hydrazide functional group and 2,5-bis(2-methoxyethoxy) terephthaloyl hydrazide containing polyethylene glycol are dissolved in an organic solvent aqueous solution in order to make each raw material component mixed evenly and form a stable solution dispersion system. Therefore, as long as the mixing method that can make each raw material component fully mixed evenly is within the scope disclosed in the embodiment specification of the present application, such as stirring, ultrasound and other methods can be used for the mixed treatment. There are no special requirements for the dissolving conditions such as temperature and time, as long as the dissolving and mixing efficiency of components such as monomer containing hydrazide functional group and 2,5-bis(2-methoxyethoxy) terephthaloyl hydrazide containing polyethylene glycol can be improved, all can be considered and implemented.

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

[0072] It should be noted that the molecular weight of the polyethylene glycol in the 2,5-bis(2-methoxyethoxy)terephthaloylhydrazide containing polyethylene glycol in the above step S10 is 400 g / mol, 600 g / mol, and 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 hydrazide functional group is a functional group containing an acyl group (RC=O) and a hydrazine group (NH-NH2), wherein the hydrazine group can also be understood as containing an amino group.

[0075] In some embodiments, in the above step S20, the monomer solution containing aldehyde functional groups may be first added to the dispersion to react thoroughly, and then an immobilization reaction may be performed under catalyst conditions, and the covalent organic framework material may be obtained after centrifugal purification.

[0076] In some embodiments, in the above step S20, the immobilization reaction is carried out under the catalyst condition in order to accelerate the reaction rate and improve the crystallinity of the covalent organic framework material. Therefore, any catalyst that can fully immobilize the monomer solution containing aldehyde functional groups and the dispersant is within the scope disclosed in the embodiment specification of the present application, such as the catalyst can be acetic acid or scandium trifluoromethanesulfonate.

[0077] In some embodiments, in the above step S20, there is no limitation on the specific implementation method of purification. For example, the reaction product after the immobilization reaction may be subjected to multiple washing, centrifugation, drying and other treatments. The reaction product may be washed with deionized water, ethanol, methanol and other solvents; the drying may be performed at high temperature or in a frozen environment.

[0078] In some embodiments, the monomer containing an aldehyde functional group in the embodiments of the present application is preferably one of trimesic acid aldehyde, 2,6-dihydroxy-1,3,5-benzenetricarboxyaldehyde, and trialdehyde phloroglucinol.

[0079] It should be noted that these aldehyde-containing functional group monomers are used as node monomers in the preparation of COF framework materials, and react with amino-containing monomers through aldehyde groups to form a network structure connected by covalent bonds. This grid structure provides a stable skeleton and permanent porosity for COF materials. At the same time, these aldehyde-containing functional group monomers react with amino-containing functional group monomers during the preparation process to form imine bonds (C=N) or other stable covalent bonds. The formation of these covalent bonds provides a guarantee for the stability and durability of COF materials.

[0080] In some embodiments, the molar ratio of the monomer containing a hydrazide functional group, the 2,5-bis(2-methoxyethoxy)terephthaloylhydrazide containing polyethylene glycol, and the monomer containing an aldehyde 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, more preferably 3:27:20. The molar ratio of different monomers determines whether a hollow capsule is formed or affects the smoothness of the shell, as well as the pore size of the capsule, which affects the substrate transfer 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. Different volume ratios allow the capsules to have different sizes, which determines whether they can be used to immobilize large-sized nanoparticles. At the same time, capsules that are too large in size affect the substrate transfer rate and thus affect the catalytic performance.

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

[0083] In a second aspect, an embodiment of the present application provides a covalent organic framework material prepared by any method described in the first aspect for use in preparing a covalent organic framework hybrid organelle.

[0084] In a third aspect, an embodiment of the present application provides a method for preparing the covalent organic framework hybrid organelle according to the second aspect, the method comprising:

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

[0086] S40, after resuspending and emulsifying the nanoparticles, coenzyme factors and biological enzymes in an aqueous solution, adding a monomer solution containing aldehyde functional groups, carrying out an immobilization reaction under the condition of scandium trifluoromethanesulfonate as a catalyst, and obtaining a covalent organic framework hybrid organelle after centrifugation and purification.

[0087] The present application can utilize the covalent organic framework material COFcap-2 to co-immobilize various enzymes, nanoparticles and coenzyme factors, and the prepared covalent organic framework hybrid organelle electroenzymatic catalysis generates various chiral amines, with higher catalytic efficiency, more excellent stability and recyclability than pure enzyme catalytic system (nitrogenase, transaminase, diaphorase, L-alanine dehydrogenase). The covalent organic framework co-immobilizes the hybrid organelles of biological and non-biological elements, and since various elements are co-fixed inside the hybrid organelles, the spatial distance can be shortened, and the cascade catalytic reaction can be effectively promoted, and energy efficient utilization is achieved, as well as efficient conversion from cheap energy to high value-added products is achieved. In addition, the tolerance of enzymes to adverse environments is effectively improved, and a protective effect is achieved, while having excellent recyclability.

[0088] It should be noted that the monomer containing the aldehyde functional group and the hydrazide functional group are dissolved in the organic solvent aqueous solution in order to make each raw material component mix uniformly and form a stable solution dispersion system. Therefore, as long as the mixing mode that can make each raw material component fully mix uniformly is in the scope disclosed in the present application embodiment specification, such as can but not only stirring, ultrasonic and other modes carry out the mixed treatment. The conditions of dissolution such as temperature and time etc. also do not have any special requirements, as long as the dissolution and mixing efficiency of components such as the monomer containing the aldehyde functional group and the monomer containing the hydrazide functional group can be improved, all can be considered and implemented.

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

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

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

[0092] It should be noted that the hydrazide functional group is a functional group containing an acyl group (RC=O) and a hydrazine group (NH-NH2), wherein the hydrazine group can also be understood as containing 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 aldehyde functional groups, and carry out an immobilization reaction under the condition of scandium trifluoromethanesulfonate as a catalyst. After centrifugation and purification, the covalent organic framework hybrid organelle is obtained.

[0094] In some embodiments, in the above step S40, there is no limitation on the specific implementation method of purification. For example, the reaction product after the immobilization reaction may be subjected to multiple washing, centrifugation, drying and other treatments. The reaction product may be washed with deionized water, ethanol, methanol and other solvents; the drying may 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 functional group to the monomer containing a hydrazide functional group is preferably 2:3.

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

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

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

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

[0102] The covalent organic framework prepared by the present invention immobilizes hybrid organelles of various biological and non-biological elements, which can effectively improve the tolerance of biological elements to adverse environments, achieve a protective effect, integrate the catalytic functions of various elements, and achieve iterative upgrading of catalytic functions.

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

[0104] Example 1

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

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

[0107] Trisaldehyde (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 trifluoromethanesulfonate aqueous solution (1.0 g / mL) was added. The mixture was vigorously stirred 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 morphological characteristics of the covalent organic framework material prepared in Example 1, the present application example tests and characterizes the covalent organic framework material prepared in Example 1. The morphology and crystallinity of COFcap-2 were observed by transmission electron microscopy (TEM), gas adsorption instrument, and powder X-ray diffraction (PXRD). Figure 1 This is a schematic diagram of the structure of the covalent organic framework material COFs provided in this application. Figure 2 Transmission electron microscopy image (a), powder X-ray diffraction image (b), gas adsorption image (c) and shell thickness schematic diagram (d) of COFcap-2 formed with different monomer ratios.

[0109] according to Figure 2 It can be seen that COFcap-2 with different monomer ratios, including DTH-400:DTH (0:30, 1:29, 3:27, 5:25, 10:20) all have good crystallinity, because the PEG chain can be properly cross-linked in the COF skeleton without forming PEG chain entanglement, but the PXRD spectrum of DTH-400:DTH (20:10, 30:0) has amorphous and low BET surface area. In addition, the introduction of DTH-400 promotes the formation of hollow COF capsules, and the increase in the proportion of DTH-400 leads to a gradual thinning of the capsule wall.

[0110] Example 2

[0111] This example provides a method for synthesizing COFcap-2 under different solvent ratios, and the specific implementation steps are as follows:

[0112] DTH-400 (0.003 mmol) and DTH (0.027 mmol) were added to a solvent system with different ratios, wherein water: chloroform was 0.5:7.5, 1.0:7.5, 2.0:7.5, and 3.0:7.5, and stirred vigorously at room temperature for 10 min (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, and then 70 μL of scandium trifluoromethanesulfonate aqueous solution (concentration of 1.0 g / mL) was added, stirred vigorously at room temperature for 50 min, and the solid was recovered by centrifugation after the reaction was completed to obtain the covalent organic framework material COFcap-2.

[0113] In order to facilitate understanding of the morphological characteristics of the covalent organic framework material prepared in Example 2, the present application example tests and characterizes the covalent organic framework material prepared in Example 2. The morphology and crystallinity of COFcap-2 were observed by transmission electron microscopy (TEM), gas adsorption instrument, and powder X-ray diffraction (PXRD). Figure 3TEM images (a) and dynamic light scattering images (b) of COFcap-2 formed with 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 with the increase of water ratio, the size of COFcap-2 capsules gradually increased.

[0115] Example 3

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

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

[0118] In order to facilitate understanding of the morphological characteristics of the immobilized product prepared in Example 3, the immobilized product prepared in Example 3 was tested and characterized in this example of the present application. 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 is able to encapsulate nanoparticles inside the material.

[0120] Example 4

[0121] This embodiment provides a COFcap-2 for coenzyme factor (NAD + ), the specific steps are as follows:

[0122] DTH-400 (0.003 mmol), DTH (0.027 mmol), NAD +(5.0 mg) was added to a 50 mL bottle of 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 solution, and the solution was added to the above solution, and then 70 μL of scandium trifluoromethanesulfonate 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 immobilized product.

[0123] In order to understand the morphological characteristics of the immobilized product prepared in Example 4, the present invention tests and characterizes the immobilized product prepared in Example 4. Characterization of NAD by X-ray Photoelectron Spectroscopy (XPS) + Package condition and NAD test + of leakage. Figure 5 The X-ray photoelectron spectroscopy image (a) and NAD of the product prepared in Example 4 + Leaked image (b).

[0124] according to Figure 5 It can be seen that COFcap-2 successfully encapsulates NAD + And it has a lower leakage rate (less than 1.3%).

[0125] Example 5

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

[0127] A total molar amount of 0.03 mmol, but different ratios of DTH-400:DTH (0:30, 1:29, 3:27, 5:25, 10:20), and 6 mg of lipase were added to a 50 mL bottle of 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 solution, and the solution was added to the above solution, and then 10 μL of scandium trifluoromethanesulfonate aqueous solution (1.0 g / mL) was added, and stirred vigorously at room temperature for 50 min. After the reaction was completed, the product was collected by centrifugation at a speed of 5500 rpm, washed with deionized water 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 run was collected and the protein concentration was quantified following the standard protocol of Bradford assay, and the residual enzyme activity of free enzyme and immobilized Lipase@COFcap-2 was determined simultaneously. Figure 6The enzyme loading (a) and enzyme activity (b) of Lipase◎COFcap-2 formed with different monomer ratios.

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

[0130] Example 6

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

[0132] DTH-400 (0.003 mmol), DTH (0.027 mmol), 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 (speed> 600 rpm). TB (3.3 mg, 0.02 mmol) was dissolved in 1.0 mL chloroform and 0.1 mL acetonitrile solution, and the solution was added to the above solution, and then 10 μL of scandium trifluoromethanesulfonate aqueous solution (1.0 g / mL) was added, and stirred vigorously at room temperature for 50 min. After the reaction was completed, the product was collected by centrifugation at a speed of 5500 rpm, washed with deionized water 3 times, and dried at room temperature to obtain GluDH◎COFcap-2 formed by different GluDH addition amounts.

[0133] After the encapsulation and washing process, the supernatant of each run was collected and the protein concentration was quantified following the standard protocol of Bradford assay, and the residual enzyme activity of free enzyme and immobilized GluDH◎COFcap-2 was determined simultaneously. Figure 7 The enzyme loading (a) and enzyme activity (b) of GluDH◎COFcap-2.

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

[0135] Example 7

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

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

[0138] After the encapsulation and washing process, the supernatant of each run was collected and the protein concentration was quantified following the standard protocol of Bradford assay, and the residual enzyme activity of free enzyme and immobilized GCDH◎COFcap-2 was determined simultaneously. 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 GCDH addition amount is 0.50 mg, it has the optimal enzyme activity, enzyme loading amount and enzyme loading rate.

[0140] Example 8

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

[0142] DTH-400 (0.003 mmol), DTH (0.027 mmol), ω-TA (added amount of 0.25 mg, 0.50 mg, 1.00 mg) were added to a 50 mL bottle of 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 solution, and the solution was added to the above solution, and then 10 μL of scandium trifluoromethanesulfonate aqueous solution (1.0 g / mL) was added, and stirred vigorously at room temperature for 50 min. After the reaction was completed, the product was collected by centrifugation at a speed of 5500 rpm, washed 3 times with deionized water, and dried at room temperature to obtain ω-TA◎COFcap-2 formed by different ω-TA addition amounts.

[0143] After the encapsulation and washing process, the supernatant of each run was collected and the protein concentration was quantified following the standard protocol of Bradford assay, and the residual enzyme activity of free enzyme and immobilized ω-TA◎COFcap-2 was determined simultaneously. Fig. 9 The enzyme loading (a) and enzyme activity (b) of ω-TA◎COFcap-2.

[0144] according to Fig. 9 It can be seen that when the ω-TA addition amount is 0.50 mg, it has the optimal enzyme activity, enzyme loading amount and enzyme loading rate.

[0145] Example 9

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

[0147] GCDH was labeled with FITC fluorescein to obtain FITC-GCDH, GluDH was labeled with RhB fluorescein to obtain RhB-GluDH, and ω-TA was labeled with DAPI fluorescein 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 at room temperature (speed> 600 rpm) for 10 min. TB (3.3 mg, 0.02 mmol) was dissolved in 1.0 mL chloroform and 0.1 mL acetonitrile solution, and the solution was added to the above solution, and then 10 μL of scandium trifluoromethanesulfonate aqueous solution (1.0 g / mL) was added, and stirred vigorously at room temperature for 50 min. After the reaction is completed, the solid is recovered by centrifugation to obtain the co-immobilized multi-enzyme biocatalyst.

[0148] The co-fixation of COFcap-2 to FITC-GCDH, RhB-GluDH and DAPI-ω-TA was observed by confocal laser scanning microscope (CLSM). Fig.10 This is a laser confocal microscopy image of COFcap-2 co-immobilizing GluDH, GCDH and ω-TA.

[0149] according to Fig.10 It can be seen that COFcap-2 uniformly fixed 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 an enzyme activity test of Lipase and Lipase◎COFcap-2, and 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) and diluted to a final volume of 3.0 mL. The reaction kinetics of free lipase were then measured at 402 nm in a thermostatically stirred UV-Vis spectrophotometer. The activity of Lipase◎COFcap-2 was determined by mixing Lipase◎COFcap-2 material containing 0.3 mg of lipase with 100 μL of p-NPA solution and then diluting to 3.0 mL with PB buffer. The activity was then determined by UV-Vis using the same method as for the free enzyme.

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

[0154] Embodiment 11

[0155] This example provides an enzyme activity test for GCDH and GCDH◎COFcap-2. The specific implementation steps are as follows: free GCDH and GCDH◎COFcap-2 containing the same mass of GCDH are added to 3 mL PB buffer (50 mM, pH = 7.0), 0.16 mM NAD + The enzyme activity was determined by measuring the increase in absorbance at 340 nm in a UV-visible spectrophotometer with constant temperature stirring.

[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: Enzyme activity test 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 are added to a mixed solution of 3 mL PB buffer (50 mM, pH = 7.0), 0.16 mM NADH, 5 mM 2-ketoglutaric acid and 100 mM NH4Cl. The enzyme activity is determined by measuring the decrease in absorbance at 340 nm in a UV-visible spectrophotometer with constant temperature stirring.

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

[0159] Embodiment 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] Free ω-TA or ω-TA◎COFcap-2 containing the same amount of ω-TA was added to 9.8 mL of PB buffer (50 mM, pH = 7.0) containing glutamic acid (20 mM) and cofactor pyridoxal 5'-phosphate monohydrate (PLP, 1.0 mM). Then, 200 μL of 4-phenyl-2-butanone solution (20 mg / mL in DMSO) 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 (2×500 μL). The organic layer was separated by centrifugation (3 min, 8000 rpm) and finally dried by adding Na2SO4. After conventional derivatization of the samples with 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 spectrometry (GC-MS) or high performance liquid chromatography (HPLC).

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

[0163] Embodiment 14

[0164] This embodiment provides a test of the ammonia production performance 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 working electrodes, and Ag / AgCl (saturated KCl) and Pt plates were used as reference and counter electrodes, respectively. The electrolyte was a 0.1 M Na2SO4 aqueous solution. Before each NRR process, the electrochemical device was first bubbled with N2 gas continuously for at least 1 h. Then, the gas was continuously bubbled throughout the electrochemical reaction, and constant potential measurements were performed at a series of applied potentials, including -0.2, -0.3, -0.4, -0.5, and -0.6 V vs Ag / AgCl, when the reaction was carried out at room temperature for 2 h. Fig.11 It is the ammonia production performance of Au◎COFcap-2.

[0166] according to Fig.11 It can be seen that Au◎COFcap-2 has the highest ammonia yield at a voltage of -0.4 V vs Ag / AgCl, reaching 6.5 mM h –1 g –1 , at a voltage of -0.3V vs Ag / AgCl, it has the highest Faradaic efficiency of 45%.

[0167] Embodiment 15

[0168] This embodiment provides a method for co-immobilizing multiple chemical catalysts 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 of 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 solution, and the solution was added to the above solution, and then 10 μL of scandium trifluoromethanesulfonate 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, and 0.2 wt% Nafion solution was added to prepare 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 generate chiral amines. Fig.12 The catalyst◎COFcap-2 has the ability to catalytically convert nitrogen into chiral amines.

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

[0171] Example 16

[0172] The effect of different biocatalyst concentrations of catalyst◎COFcap-2 on the cascade catalytic activity is shown in the following specific implementation steps:

[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 addition 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 of 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 solution, and the solution was added to the above solution, and then 10 μL of scandium trifluoromethanesulfonate aqueous solution (1.0 g / mL) was added, and stirred vigorously at room temperature for 50 min. After the reaction is completed, the solid is recovered by centrifugation, and 0.2 wt% Nafion solution is added to prepare catalyst◎COFcap-2 paste, which is coated on the surface of carbon paper to prepare a working electrode for catalytic conversion of nitrogen to generate chiral amines. Fig.13 The performance of catalyst◎COFcap-2 formed by different biocatalyst ratios in catalyzing the conversion of nitrogen into chiral amines.

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

[0175] Embodiment 17

[0176] The catalytic performance test of catalyst◎COFcap-2 on different catalytic substrates is carried out in the following specific steps:

[0177] In vitro catalytic system synthesis of chiral amine drug molecules was performed at a constant potential of -0.4V vs Ag / AgCl at 30°C in an H-shaped dual-chamber bioelectrochemical reactor. A paste of catalyst◎COFcap-2 containing 0.2wt% Nafion was applied on carbon paper and used as a working electrode before air drying. The electrolyte was a 0.1M Na2SO4 aqueous solution containing 1mM PLP, 5mM glucose, 1mM 2-ketoglutaric acid, 1mM ketone substrate and 1% DMSO. Constant potential measurements were performed at -0.4V vs Ag / AgCl for 24h at room temperature. The reaction mixture was extracted with ethyl acetate. The organic layer was separated by centrifugation and finally dried over Na2SO4. After conventional derivatization of the samples with 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 spectrometry (GC-MS) or high performance liquid chromatography (HPLC). Fig.14 The performance of catalyst◎COFcap-2 in catalyzing the conversion of nitrogen into different chiral amines.

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

[0179] Embodiment 18

[0180] The recyclability 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 rinsed twice with phosphate buffer, and the reaction was carried out again according to the reaction system of Example 17. After the reaction, samples were taken to measure the conversion rate and residual activity of each batch. The ratio of the residual enzyme activity of the fifteenth reaction to the first reaction was the residual enzyme activity after repeated use for fifteen times, so as to examine the catalytic efficiency of the catalyst◎COFcap-2 co-immobilized catalyst and the change of the residual activity with the increase of the number of uses. Fig.15 It is the recyclability of catalyst◎COFcap-2.

[0182] Results Fig.15 The relative yield of the co-immobilized catalyst ◎COFcap-2 is still higher than 60% after being reused fifteen times. The experimental difficulty of the entire catalytic system is low, the overall conversion efficiency is high, and the cost can be further reduced.

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

[0184] COFcap-2 combines chemical catalysis (NRR) and biocatalysis (enzyme cascade) to achieve N2 fixation to homochiral amines, such as the drug compound (R)-sitagliptin, a blockbuster drug for the treatment of type 2 diabetes The active ingredient in.

[0185] The 11 substrates in the examples of this application verified the performance of COFcap-2. Each substrate had good catalytic performance and excellent enantioselectivity, such as 2-amino-4-phenylbutane (62 μM·h -1 The performance of COFcap-2 was better than that of the reported homogeneous catalysts and maintained good relative yields after 15 cycles.

[0186] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A method for preparing a covalent organic framework material, characterized in that: include: Dissolving a monomer containing a hydrazide functional group and 2,5-bis(2-methoxyethoxy)terephthaloylhydrazide containing polyethylene glycol in an organic solvent aqueous solution to form a dispersion; A monomer solution containing aldehyde functional groups is added to the dispersion, an immobilization reaction is carried out under catalyst conditions, and a covalent organic framework material is obtained after centrifugal purification.

2. The method according to claim 1, characterized in that The monomer containing a hydrazide functional group is at least one of 2,5-diethoxybenzene-1,4-di(formyl hydrazide), 2,5-bis(2-methoxyethoxy)terephthaloyl hydrazide, 2,5-bis(but-3-ene-1-oxy)terephthaloyl hydrazide, and 2-(but-3-ene-1-oxy)-5-(2-methoxyethoxy)terephthaloyl hydrazide; And / or, the molecular weight of the polyethylene glycol in the 2,5-bis(2-methoxyethoxy)terephthaloyl hydrazide containing polyethylene glycol is at least one of 400 g / mol, 600 g / mol, and 800 g / mol; And / or, the monomer containing aldehyde functional group is at least one of trimesaldehyde, 2,6-dihydroxy-1,3,5-benzenetricarbaldehyde and trialdehyde phloroglucinol; And / or, 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, and a compound containing hydroxyl functional groups.

3. The method according to claim 1, characterized in that: The molar ratio of the monomer containing hydrazide functional group, the 2,5-bis(2-methoxyethoxy)terephthaloyl hydrazide containing polyethylene glycol and the monomer containing aldehyde functional group 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 to organic solvent in the organic solvent aqueous solution is 0.5:7.5, 1.0:7.5, 2.0:7.5, 3.0:7.

5.

4. The method according to claim 3, characterized in that The molar ratio of the monomer containing hydrazide functional group, the 2,5-bis(2-methoxyethoxy)terephthaloyl hydrazide containing polyethylene glycol and the monomer containing aldehyde functional group is 3:27:20; and / or, the volume ratio of water to 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 trifluoromethanesulfonate.

5. Use of a covalent organic framework material prepared according to any one of claims 1 to 4 in preparing a covalent organic framework hybrid organelle.

6. A method for preparing a covalent organic framework hybrid organelle according to claim 5, characterized in that: include: Dissolving a monomer containing an aldehyde functional group and a monomer containing a hydrazide functional group in an organic solvent aqueous solution to form a dispersion; After the nanoparticles, coenzyme factors and biological enzymes are resuspended and emulsified in an aqueous solution, a monomer solution containing an aldehyde functional group is added, and an immobilization reaction is carried out under the condition that scandium trifluoromethanesulfonate is used as a catalyst. After centrifugation and purification, a covalent organic framework hybrid organelle is obtained.

7. The method according to claim 6, characterized in that The monomer containing an aldehyde functional group is at least one of trimesaldehyde, 2,6-dihydroxy-1,3,5-benzenetricarbaldehyde, and trialdehyde phloroglucinol; And / or, the monomer containing a hydrazide functional group is at least one of 2,5-bis(2-methoxyethoxy)terephthaloyl hydrazide, 2,5-bis(but-3-ene-1-oxy)terephthaloyl hydrazide, and 2-(but-3-ene-1-oxy)-5-(2-methoxyethoxy)terephthaloyl hydrazide; And / or, the organic solvent is chloroform; And / or, the nanoparticles are at least one of gold nanoparticles, iron nanoparticles, and copper nanoparticles; And / or, the biological enzyme is at least one of transaminase, glucose dehydrogenase, glutamate dehydrogenase, leucine dehydrogenase, glucose oxidase, catalase, and lipase.

8. The method according to claim 7, characterized in that 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 to 40°C; And / or, the added amount of the biological enzyme is 0.25-1.0 mg; And / or, the amount of scandium trifluoromethanesulfonate added is 10 to 70 μL; And / or, the added amount of the coenzyme factor is 5.0 mg.

9. The method according to claim 8, characterized in that The added amount of the biological enzyme is 0.5 mg; And / or, the temperature of the immobilization reaction is 20-30°C.

10. Use of the covalent organic framework hybrid organelle prepared by the method according to any one of claims 6 to 9 in producing active pharmaceutical ingredients or pharmaceutical intermediates.

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