MOF (Metal Organic Framework) hybrid hydrogel microbead for immobilizing double enzymes as well as preparation method and application of MOF hybrid hydrogel microbead

Abstract

The invention relates to the technical field of bioengineering, and particularly discloses MOF (Metal Organic Framework) hybrid hydrogel microspheres for immobilizing double enzymes as well as a preparation method and application of the MOF hybrid hydrogel microspheres. The double enzymes in the double-enzyme-immobilized MOF hybrid hydrogel microbead are a first enzyme and a second enzyme which form a cascade catalytic reaction, and the double-enzyme-immobilized MOF hybrid hydrogel microbead has a spatial layered structure; the spatial layered structure comprises an MOF (Metal Organic Framework) embedded with a first enzyme and hydrogel which is distributed around the MOF and is loaded with a second enzyme. In the MOF hybrid hydrogel microbead for immobilizing the double enzymes, the first enzyme is encapsulated in a rigid MOF lattice, the conformation of the first enzyme is effectively immobilized, and the high temperature resistance, the pH resistance and the organic solvent resistance are remarkably enhanced; the second enzyme is distributed in the hydrogel matrix around the MOF, and the hydrogel network also provides a mild protection environment for the second enzyme. The spatial layered structure effectively isolates the two enzymes, and avoids direct contact and potential mutual inhibition between the two enzymes.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a MOF hybrid hydrogel microbead with immobilized dual enzymes, its preparation method, and its application. Background Technology

[0002] Enzymes, as highly efficient and specific biocatalysts, play a vital role in fields such as medicine, food, chemical industry, and environmental monitoring. However, free enzymes often face problems in practical applications, such as poor stability, difficulty in recycling, and sensitivity to environmental factors (e.g., temperature, pH), which greatly limit their industrial application. To overcome these shortcomings, enzyme immobilization technology has emerged.

[0003] In recent years, multi-enzyme cascade catalytic systems have attracted widespread attention due to their ability to mimic complex metabolic networks in vivo, achieve one-pot multi-step reactions, reduce the separation and purification steps of intermediate products, and improve overall reaction efficiency. However, the optimal working conditions of different enzymes may vary, and non-specific interactions or inhibition may occur between enzyme molecules or between enzymes and substrates / products within the same space, leading to a decrease in catalytic efficiency. Therefore, developing carriers and methods that can achieve spatially ordered enzyme arrangement (i.e., partitioned immobilization) is crucial. Summary of the Invention

[0004] The present invention aims to solve at least one of the aforementioned technical problems existing in the prior art.

[0005] Therefore, the first objective of this invention is to provide a MOF hybrid hydrogel microsphere with immobilized dual enzymes; the second objective of this invention is to provide a method for preparing such MOF hybrid hydrogel microspheres with immobilized dual enzymes; and the third objective of this invention is to provide the application of such MOF hybrid hydrogel microspheres with immobilized dual enzymes in the fields of constructing biosensors, producing pharmaceutical intermediates, synthesizing fine chemicals, and degrading environmental pollutants.

[0006] The inventive concept of this invention is as follows: Metal-organic frameworks (MOFs) are a class of porous crystalline materials formed by the self-assembly of metal ions / clusters and organic ligands. Due to their high specific surface area, tunable pore structure, and good chemical stability, MOFs are considered highly promising enzyme immobilization carriers. In-situ encapsulation, where enzymes are embedded as guest molecules during MOF synthesis, can achieve efficient enzyme encapsulation under mild conditions, providing a robust "armor" for the enzyme molecules and effectively preventing leaching and denaturation. Hydrogels, especially natural polymer hydrogels such as alginate, are widely used for immobilizing biomolecules due to their excellent biocompatibility, high water content, and mild gelation conditions. This invention combines the rigid porous structure of MOFs with the flexible network of hydrogels to construct MOF-hydrogel hybrid materials, systematically achieving a two-enzyme layered immobilization strategy while retaining the advantages of both.

[0007] Specifically, the MOF hybrid hydrogel microbeads immobilized with dual enzymes of this invention have a spatially layered structure consisting of an MOF encapsulating the first enzyme and a hydrogel loaded with the second enzyme distributed around the MOF. The first enzyme is encapsulated in a rigid MOF lattice, effectively immobilizing its conformation and significantly enhancing its resistance to high temperatures, pH, and organic solvents. The second enzyme is distributed in the hydrogel matrix surrounding the first enzyme@MOF complex, and the hydrogel network also provides a mild protective environment for the second enzyme. This spatially layered structure achieves spatial partitioning of the dual enzymes, effectively avoiding mutual interference between enzymes, enhancing the efficiency of cascade reactions, and significantly improving the enzyme's thermal stability, operational stability, storage stability, and recyclability. It effectively addresses the problems of small size of enzyme particles immobilized by a single MOF and low enzyme loading capacity of the hydrogel, simplifies post-processing procedures during application, and overcomes the limitations of traditional enzyme co-immobilization methods.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides MOF hybrid hydrogel microbeads with immobilized dual enzymes, wherein the dual enzymes are a first enzyme and a second enzyme constituting a cascade catalytic reaction, and the MOF hybrid hydrogel microbeads with immobilized dual enzymes have a spatially layered structure; the spatially layered structure includes an MOF encapsulating the first enzyme and a hydrogel loaded with the second enzyme distributed around the MOF.

[0009] In this invention, the MOF hybrid hydrogel microbeads immobilized with two enzymes have a spatially layered structure. This spatially layered structure realizes the spatial partitioning of the two enzymes, effectively avoids mutual interference between enzymes, enhances the efficiency of cascade reactions, and significantly improves the thermal stability, operational stability, storage stability, and recyclability of the enzymes.

[0010] Preferably, the stability of the first enzyme is lower than that of the second enzyme. The MOF rigid framework can isolate the enzyme from damage caused by extreme pH, organic solvents, proteases, etc., and prolong the enzyme's lifespan. Therefore, it is used for the first enzyme, which has poor stability in a two-enzyme combination.

[0011] Preferably, the particle size of the MOF hybrid hydrogel microbeads with immobilized dual enzymes is 1~3 mm.

[0012] In this invention, the MOF hybrid hydrogel microbeads immobilized with dual enzymes are uniform in size, with a particle size of 1-3 mm, and are easy to separate from the reaction system by filtration or sedimentation, thus realizing easy recovery and recycling of the enzymes and greatly reducing application costs.

[0013] Preferably, the first enzyme is selected from one of aldehyde-ketone reductase, alcohol dehydrogenase, glucose oxidase, transaminase, amylase, peroxidase, pyruvate dehydrogenase, pectinase, carbon dioxide dehydrogenase, monooxygenase, urease, cholinesterase, and lipase.

[0014] Preferably, the second enzyme is selected from one of formate dehydrogenase, catalase, horseradish peroxidase, glutamate dehydrogenase, saccharifying enzyme, laccase, lactate dehydrogenase, cellulase, formate dehydrogenase, glucose dehydrogenase, peroxidase, and esterase.

[0015] Preferably, the first enzyme and the second enzyme comprise at least one pair of the following enzyme combinations: aldehyde-ketone reductase and formate dehydrogenase, alcohol dehydrogenase and formate dehydrogenase, glucose oxidase and catalase, glucose oxidase and horseradish peroxidase, transaminase and glutamate dehydrogenase, amylase and saccharifying enzyme, peroxidase and laccase, pyruvate dehydrogenase and lactate dehydrogenase, pectinase and cellulase, carbon dioxide dehydrogenase and formate dehydrogenase, monooxygenase and glucose dehydrogenase, urease and glutamate dehydrogenase, cholinesterase and peroxidase, lipase and esterase.

[0016] Secondly, the method for preparing the MOF hybrid hydrogel microbeads with immobilized dual enzymes as described in the first aspect of the present invention includes the following steps: S1: The first enzyme is encapsulated within an MOF to prepare a first enzyme@MOF complex; the first enzyme@MOF complex is a core-shell structure with the first enzyme as the core and the MOF as the shell, and the first enzyme is encapsulated within the MOF shell. S2: The first enzyme@MOF complex, the second enzyme, and the hydrogel components are dispersed in the medium to form a hybrid hydrogel precursor solution; S3: Crosslink the hybrid hydrogel precursor solution with a crosslinking agent to form MOF hybrid hydrogel microbeads with immobilized dual enzymes.

[0017] In this invention, the method for preparing MOF hybrid hydrogel microbeads with immobilized dual enzymes provided by the inventors mainly consists of three stages: the preparation stage of the first enzyme@MOF complex, the preparation stage of the hybrid hydrogel precursor solution, and the cross-linking and molding stage of the hybrid hydrogel microbeads. In the first stage, MOFs are used to in-situ embed the first enzyme. During the MOF lattice formation process, the first enzyme is encapsulated in-situ within pores or cavities, forming a nanoscale first enzyme@MOF complex. The embedding efficiency can reach 60%~99%, avoiding enzyme molecule aggregation or degradation. Mild synthesis conditions (room temperature, aqueous solution) can maximize enzyme activity. The rigid framework of MOFs can isolate the enzyme from extreme pH, organic solvents, proteases, etc., extending the enzyme's lifespan. It can be used to immobilize enzymes with poor stability in dual enzymes. The high specific surface area and abundant pore structure of MOFs facilitate rapid substrate / product diffusion and reduce mass transfer limitations. In the second stage, the first enzyme@MOF, the second enzyme, and the hydrogel components are mixed to obtain the hybrid hydrogel precursor. In the third stage, a cross-linking agent is used to cross-link the hybrid hydrogel precursor, forming uniform spherical hybrid hydrogel microbeads. The prepared hybrid hydrogel microbeads have a particle size of 1-3 mm, exhibit good stability and mechanical properties, are easily separated from the reaction system by filtration or sedimentation, and retain the activity of both enzymes (enzyme activity recovery rate is higher than 90%), enabling easy enzyme recovery and multiple recycling, greatly reducing application costs. Compared with systems using MOF to directly embed mixed enzymes in situ or using composite polymers to directly embed mixed enzymes in situ, the MOF hybrid hydrogel microbead immobilization system prepared in this invention effectively isolates the two enzymes, avoiding direct contact and potential mutual inhibition between them, shortening the diffusion distance of intermediate products, and greatly improving the overall efficiency of the cascade reaction.

[0018] Preferably, the MOF is at least one of ZIF-8, ZIF-90, ZPF-2, ZIF-67, and ZIF-L; more preferably, the MOF is ZIF-8 and / or ZIF-90.

[0019] Preferably, the specific method for encapsulating the first enzyme within the MOF is to add a metal salt solution of the MOF to a mixed solution of the organic ligand solution of the MOF and the first enzyme for in-situ self-assembly reaction. After the reaction is completed, the product is obtained by centrifugation, and the product is freeze-dried to obtain the first enzyme@MOF complex.

[0020] Preferably, the first enzyme is in the form of an enzyme preparation, a solution containing an enzyme preparation, or a crude enzyme solution obtained by treating enzyme-containing sludge.

[0021] Preferably, the method for treating the enzyme-containing sludge is to disperse the enzyme-containing sludge in a buffer solution with a pH of 6.8 to 7.2, ultrasonically break it up, centrifuge it, and then take the supernatant to obtain a crude enzyme solution.

[0022] Preferably, the protein concentration of the crude enzyme solution is 2-12 mg / mL; more preferably, the protein concentration of the crude enzyme solution is 3-7 mg / mL; and even more preferably, the protein concentration of the crude enzyme solution is 4-6 mg / mL.

[0023] Preferably, the concentration of the enzyme preparation in the solution containing the enzyme preparation is 0.5~50 mg / mL; more preferably, the concentration of the enzyme preparation in the solution containing the enzyme preparation is 1~30 mg / mL; even more preferably, the concentration of the enzyme preparation in the solution containing the enzyme preparation is 5~20 mg / mL.

[0024] Preferably, the metal salt of the MOF includes one of zinc salt and cobalt salt.

[0025] Preferably, the zinc salt includes at least one of zinc nitrate, zinc acetate, zinc chloride, zinc sulfate, and zinc acetate.

[0026] Preferably, the cobalt salt includes at least one of cobalt nitrate and cobalt acetate.

[0027] Preferably, the organic ligand of the MOF includes one of 2-methylimidazole, imidazole-2-carboxaldehyde, and 4-chloro-5-chloromethylimidazole.

[0028] Preferably, the molar ratio of the metal salt of the MOF to the organic ligand of the MOF is (1:4) to (1:40); more preferably, the molar ratio of the metal salt of the MOF to the organic ligand of the MOF is (1:6) to (1:30); even more preferably, the molar ratio of the metal salt of the MOF to the organic ligand of the MOF is (1:8) to (1:20).

[0029] Preferably, the molar concentration of the metal salt in the MOF metal salt solution is 0.01~1 mol / L; more preferably, the molar concentration of the metal salt is 0.02~0.5 mol / L; even more preferably, the molar concentration of the metal salt is 0.02~0.4 mol / L. Preferably, the molar concentration of the organic ligand in the organic ligand solution of the MOF is 0.05~2 mol / L; more preferably, the molar concentration of the organic ligand is 0.07~1.5 mol / L; even more preferably, the molar concentration of the organic ligand is 0.08~1.3 mol / L.

[0030] Preferably, the amount of the first enzyme added is 30-250 mg; more preferably, the amount of the first enzyme added is 50-200 mg; and even more preferably, 100-180 mg. When the first enzyme is in the form of a crude enzyme solution, the concentration of the first enzyme is calculated based on the enzyme protein content in the crude enzyme solution.

[0031] Preferably, the in-situ self-assembly reaction temperature is 4~30℃; more preferably, the in-situ self-assembly reaction temperature is 10~28℃; even more preferably, the in-situ self-assembly reaction temperature is 20~26℃; and even more preferably, the in-situ self-assembly reaction temperature is 25℃.

[0032] Preferably, the stirring time for the in-situ self-assembly reaction is 10-60 minutes; more preferably, the stirring time for the in-situ self-assembly reaction is 20-40 minutes; and even more preferably, the stirring time for the in-situ self-assembly reaction is 25-35 minutes.

[0033] Preferably, the obtained precipitate is washed with deionized water and then freeze-dried; more preferably, the washing is performed 2 to 5 times, and even more preferably, the washing is performed 3 times.

[0034] Preferably, the second enzyme in S2 is in the form of an enzyme preparation, a solution containing an enzyme preparation, or a crude enzyme solution obtained by treating enzyme-containing sludge.

[0035] Preferably, the method for treating the enzyme-containing sludge is to disperse the enzyme-containing sludge in a buffer solution with a pH of 6.5 to 7.5, ultrasonically break it up, centrifuge it, and then take the supernatant to obtain a crude enzyme solution.

[0036] Preferably, the medium in S2 is an aqueous medium; more preferably, the aqueous medium is deionized water or a buffer solution; even more preferably, the pH of the buffer solution is 6.5 to 7.5.

[0037] Preferably, the hydrogel component includes alginate and auxiliary polymer.

[0038] In this invention, alginate is used as a mixed auxiliary polymer. This auxiliary polymer can form an interpenetrating or semi-interpenetrating network structure with alginate, thereby improving the mechanical properties of the hydrogel material and enhancing the stability of the mixed hydrogel. This avoids the problems of low mechanical strength, structural instability, and easy degradation in specific buffer solutions inherent in purely ionicly cross-linked alginate gels. The auxiliary polymers used in this invention are all water-soluble polymers with good biocompatibility and chemical stability, effectively maintaining the biological activity of the first enzyme@MOF complex and the second enzyme.

[0039] Preferably, the alginate is sodium alginate.

[0040] Preferably, the auxiliary polymer includes at least one of chitosan (CS), gelatin, starch, carboxymethyl cellulose, hydroxypropyl methyl cellulose, pectin, polyvinyl alcohol (PVA), polyethylene glycol, polyacrylamide, and polylactic acid.

[0041] In some embodiments of the present invention, when the auxiliary polymer is chitosan, the chitosan can be dispersed in the medium in S2 or in the crosslinking solution in S3.

[0042] Preferably, the concentration (w / v) of alginate in the hybrid hydrogel precursor solution is 3~50 mg / mL; more preferably, the concentration of alginate in the hybrid hydrogel precursor solution is 5~30 mg / mL; even more preferably, the concentration of alginate in the hybrid hydrogel precursor solution is 10~30 mg / mL.

[0043] Preferably, the concentration (w / v) of the auxiliary polymer in the hybrid hydrogel precursor solution is 0.1~100 mg / mL; more preferably, the concentration of the auxiliary polymer in the hybrid hydrogel precursor solution is 5~60 mg / mL; even more preferably, the concentration of the auxiliary polymer in the hybrid hydrogel precursor solution is 10~30 mg / mL.

[0044] Preferably, the concentration (w / v) of the first enzyme@MOF complex in the hybrid hydrogel precursor solution is 0.1~100 mg / mL; more preferably, the concentration of the first enzyme@MOF complex in the hybrid hydrogel precursor solution is 0.5~50 mg / mL; even more preferably, the concentration of the first enzyme@MOF complex in the hybrid hydrogel precursor solution is 1~30 mg / mL.

[0045] Preferably, the concentration of the second enzyme in the hybrid hydrogel precursor solution is 0.1~10 mg / mL; more preferably, the concentration of the second enzyme in the hybrid hydrogel precursor solution is 0.5~8 mg / mL; even more preferably, the concentration of the second enzyme in the hybrid hydrogel precursor solution is 1~5 mg / mL. When the second enzyme is in the form of a crude enzyme solution, the concentration of the second enzyme is calculated based on the enzyme protein content in the crude enzyme solution.

[0046] Preferably, the crosslinking agent in S3 includes metal ion crosslinking agents and / or chemical crosslinking agents.

[0047] Preferably, the metal ions in the metal ion crosslinking agent include Ca. 2+ Zn 2+ Mg 2+ Al 3+ Fe 2+ Fe 3+ At least one of them.

[0048] Preferably, the chemical crosslinking agent includes at least one of glutaraldehyde, glyoxal, and isocyanate.

[0049] Preferably, the specific method for reacting the hybrid hydrogel precursor solution with the crosslinking agent in S3 is to add the hybrid hydrogel precursor solution to the crosslinking solution to carry out the crosslinking reaction.

[0050] Preferably, the addition is done by uniform dripping; more preferably, the rate is 0.5~1.5 mL / min; even more preferably, the rate is 0.7~1.3 mL / min; and even more preferably, the rate is 0.8~1.2 mL / min.

[0051] Preferably, the concentration (w / v) of the metal ion crosslinking agent in the crosslinking solution in S3 is 1%-8%; more preferably, the concentration of the metal ion crosslinking agent in the crosslinking solution in S3 is 1.5%-5.5%; even more preferably, the concentration of the metal ion crosslinking agent in the crosslinking solution in S3 is 2%-5%. The unit of concentration (w / v) is g / mL.

[0052] Preferably, the concentration (w / v) of glutaraldehyde in the crosslinking solution in S3 is 0.1%-5%; more preferably, the concentration of glutaraldehyde in the crosslinking solution in S3 is 0.5%-4.5%; even more preferably, the concentration of glutaraldehyde in the crosslinking solution in S3 is 1%-2%. The unit of concentration (w / v) is g / mL.

[0053] In this invention, to enhance the stability and mechanical properties of hybrid hydrogel microspheres, a three-dimensional cross-linking network is constructed using a combination of chemical cross-linking and metal ion cross-linking. The hybrid microspheres cross-linked with glutaraldehyde exhibit significantly improved mechanical strength, swelling resistance, and acid and alkali resistance.

[0054] Preferably, the volume ratio of the hybrid hydrogel precursor solution to the crosslinking solution in S3 is (1:3) to (1:30); more preferably, the volume ratio of the hybrid hydrogel precursor solution to the crosslinking solution in S3 is (1:5) to (1:20); even more preferably, the volume ratio of the hybrid hydrogel precursor solution to the crosslinking solution in S3 is (1:8) to (1:10).

[0055] Preferably, the stirring time for the crosslinking reaction in S3 is 20 to 240 minutes; more preferably, the stirring time for the crosslinking reaction in S3 is 25 to 120 minutes; and even more preferably, the stirring time for the crosslinking reaction in S3 is 30 to 60 minutes.

[0056] Preferably, the crosslinking reaction is allowed to stand for 1 to 6 hours to ensure complete crosslinking; more preferably, the crosslinking reaction is allowed to stand for 1 to 4 hours to ensure complete crosslinking; even more preferably, the crosslinking reaction is allowed to stand for 1 to 2 hours to ensure complete crosslinking.

[0057] Thirdly, the present invention provides an application of the MOF hybrid hydrogel microspheres with immobilized dual enzymes as described in the first aspect of the present invention in the fields of constructing biosensors, producing pharmaceutical intermediates, synthesizing fine chemicals, and degrading environmental pollutants.

[0058] Preferably, the biosensor is a glucose biosensor.

[0059] Preferably, the application method of MOF hybrid hydrogel microbeads immobilized with two enzymes in a glucose biosensor is as follows: 1) MOF hybrid hydrogel beads immobilized with two enzymes were placed in phosphate buffer solutions containing chromogenic substrates and different concentrations of glucose for catalytic oxidation reaction. The two enzymes were Gox and HRP. After the reaction, the absorbance values ​​of glucose solutions of different concentrations were measured at 650 nm, and a standard curve of absorbance value-glucose concentration was plotted. 2) Add MOF hybrid hydrogel beads with immobilized dual enzymes and chromogenic substrate to the glucose phosphate buffer solution to be tested for catalytic oxidation reaction. After the reaction is completed, measure the absorbance value of the solution at 650 nm. Quantify the glucose concentration in the glucose phosphate buffer solution to be tested according to the absorbance value-glucose concentration standard curve.

[0060] Preferably, the number of MOF hybrid hydrogel microbeads immobilizing the two enzymes is 20-50; more preferably, the number of MOF hybrid hydrogel microbeads immobilizing the two enzymes is 25-45; and even more preferably, the number of MOF hybrid hydrogel microbeads immobilizing the two enzymes is 28-35.

[0061] Preferably, the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine (TMB) or o-toluidine.

[0062] Preferably, the concentration of the chromogenic substrate is 0.5~2 mM; more preferably, the concentration of the chromogenic substrate is 0.8~1.5 mM; even more preferably, the concentration of the chromogenic substrate is 1~1.2 mM.

[0063] Preferably, the catalytic oxidation time is 10-150 min; more preferably, the catalytic oxidation time is 30-120 min; and even more preferably, the catalytic oxidation time is 60-100 min.

[0064] The beneficial effects of this invention are as follows: 1. The MOF hybrid hydrogel microbeads immobilizing two enzymes of the present invention have a spatially layered structure, comprising an MOF encapsulating a first enzyme and a hydrogel loaded with a second enzyme distributed around the MOF. The first enzyme is encapsulated in a rigid MOF lattice, its conformation is effectively immobilized, and its resistance to high temperature, pH, and organic solvents is significantly enhanced. The second enzyme is distributed in the hydrogel matrix surrounding the first enzyme@MOF complex, and the hydrogel network also provides a mild protective environment for the second enzyme. This spatially layered structure effectively isolates the two enzymes, avoiding direct contact and potential mutual inhibition between them, while shortening the diffusion distance of intermediate products and greatly improving the overall efficiency of the cascade reaction.

[0065] 2. The entire preparation process of this invention is carried out under mild conditions (such as room temperature and an aqueous system), which maximizes the preservation of the enzyme's natural activity. This method has good versatility, is simple to prepare, has low equipment requirements, and can be extended to other dual-enzyme or multi-enzyme systems by changing different MOFs, hydrogel components, and enzyme combinations. It is suitable for the application of various enzyme pair systems in various fields and is suitable for large-scale production.

[0066] 3. The MOF hybrid hydrogel microspheres with immobilized dual enzymes provided by this invention are far superior to free enzymes in terms of thermal stability, operational stability, long-term storage stability and recyclability. They can be well applied in the fields of constructing biosensors (such as glucose biosensors), producing pharmaceutical intermediates, synthesizing fine chemicals and degrading environmental pollutants. Attached Figure Description

[0067] Figure 1 This is a schematic diagram of the preparation method of AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads in Example 1; Figure 2 The reaction equation for the catalytic production of (R)-6-hydroxy-8-chlorooctanoate ethyl ester by AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads; Figure 3 The absorbance-glucose concentration standard curve plotted for Example 1 of the glucose biosensor; Figure 4 The image shown is a transmission electron microscope (TEM) image of the AKR@ZIF-8 complex obtained in step 1) of Example 1. Figure 5 Optical photograph of the AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres prepared in Example 1; Figure 6 Transmission electron microscopy image of the AKR-FDH@ZIF-8 complex prepared in Comparative Example 1; Figure 7Optical photograph of AKR-FDH@SA / PVA hybrid hydrogel microbeads prepared for Comparative Example 2; Figure 8 The image shown is a transmission electron microscope (TEM) image of the GOx@ZIF-90 composite obtained in step 1) of Example 3. Figure 9 Optical photograph of GOx@ZIF-90-HRP@SA / gelatin hybrid hydrogel microbeads prepared in Example 3; Figure 10 X-ray powder diffraction patterns of the AKR@ZIF-8 composite and AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres prepared in Example 1; Figure 11 Fourier transform infrared spectra of the AKR@ZIF-8 complex, AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres obtained in step 1) of Example 1, the AKR-FDH@ZIF-8 complex prepared in Comparative Example 1, and the AKR-FDH@SA / PVA hybrid hydrogel microspheres prepared in Comparative Example 2. Figure 12 X-ray powder diffraction patterns of GOx@ZIF-90 and GOx@ZIF-90-HRP@SA / gelatin hybrid hydrogel microspheres prepared in Example 3; Figure 13 The TLC results of the reaction solution at different time points are shown in Example 1, Comparative Example 1, and Comparative Example 2 of the microbead catalytic reaction during the catalytic production of (R)-6-hydroxy-8-chlorooctanoate. Figure 14 Gas chromatogram of (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester after 4 hours of catalytic reaction of 6-carbonyl-8-chlorooctanoic acid ethyl ester in Example 1 of microbead catalysis reaction; Figure 15 The diagram shows the chiral test results of (R)-6-hydroxy-8-chlorooctanoate ethyl ester obtained by the catalytic production of the microbead catalytic reaction in Example 1. Figure 16 The graph shows the relative enzyme activity data for 5 rounds of cyclic catalysis using AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel beads. Figure 17 Figure 1 shows the storage stability test results of free AKR and AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads at 30°C. Figure 18 The figure shows the storage stability test results of FDH immobilized in free FDH and AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads at 30°C. Detailed Implementation

[0068] To enable those skilled in the art to better understand this application, the present invention will be further described in detail below with reference to embodiments. However, it should be understood that the following embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims.

[0069] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced even without certain specific details. In some embodiments, raw materials, methods, and means well-known to those skilled in the art are not described in detail, in order to highlight the main points of the present invention. In the description of the present invention, it should be noted that conditions not specifically specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0070] The raw materials used in the embodiments and comparative examples of this invention are sourced from the following sources: Bacterial sludge containing aldehyde-ketone reductase (AKR19): The recombinant bacterial cells expressing aldehyde-ketone reductase disclosed in sections

[0025] to

[0026] of CN118685463A were fermented (the fermentation method can be conventional or refer to the fermentation method in section

[0029] of CN118685463A). After centrifuging the fermented bacterial broth at 12000 rpm for 10 minutes, the wet bacterial cells obtained are the bacterial sludge containing aldehyde-ketone reductase (AKR19); in this application, AKR represents AKR19. Formate dehydrogenase (FDH)-containing bacterial sludge: The formate dehydrogenase recombinant Escherichia coli engineered bacteria disclosed in paragraphs

[0034] ~

[0035] of the instruction manual CN120210139 A are fermented (the fermentation method can be conventional or refer to the culture method of the strain in paragraph S1 of paragraph

[0036] of the instruction manual CN120210139 A). After centrifuging the fermentation broth of the formate dehydrogenase recombinant Escherichia coli obtained by culture at 12000 rpm for 10 minutes, the wet bacterial cells obtained are the formate dehydrogenase-containing bacterial sludge. Glucose oxidase (GOx): Commercially available, purchased from Beijing Solarbio Science & Technology Co., Ltd. Horseradish peroxidase (HRP): Commercially available, purchased from Beijing Solarbio Technology Co., Ltd.

[0071] The room temperature in this invention is 25°C.

[0072] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0073] Example 1 A method for preparing AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres, the process flow diagram of which is shown below. Figure 1 As shown, the specific preparation steps are as follows: 1) Preparation of AKR@ZIF-8 complex: Accurately weigh 0.8g of bacterial sludge containing aldehyde reductase, disperse it in 40mL of phosphate buffer solution (pH=7.0), sonicate to disrupt, centrifuge at 10000rpm for 5 minutes, and take the supernatant to obtain crude AKR enzyme solution (protein concentration: 4mg / mL). Weigh 367 mg of anhydrous zinc acetate and dissolve it in 100 mL of deionized water to obtain a metal salt solution (concentration of 20 mM). Weigh 657 mg of 2-methylimidazole and dissolve it in 100 mL of deionized water to obtain an organic ligand solution (concentration 80 mM). Under room temperature and magnetic stirring, 40 mL of crude AKR enzyme solution and 100 mL of organic ligand solution were mixed evenly to obtain a mixed solution. 100 mL of metal salt solution was quickly added to the mixed solution, and the reaction was continued to be stirred for 30 min. After the reaction was completed, the product was centrifuged at 10000 rpm for 5 min and washed 3 times with deionized water. Finally, the product was freeze-dried to obtain AKR@ZIF-8 complex powder.

[0074] 2) Preparation of hybrid hydrogel precursor solution: Weigh 2g of bacterial sludge containing formate dehydrogenase (FDH), disperse it in 100mL of phosphate buffer solution (pH=7.0), sonicate to break it up, centrifuge at 10000rpm for 5 minutes, and take the supernatant to obtain crude FDH enzyme solution (protein concentration: 4mg / mL). Weigh 0.2 g sodium alginate and 0.2 g polyvinyl alcohol, add them to 10 mL deionized water, heat and stir in a 60 °C water bath until completely dissolved, and cool to room temperature; then add 100 mg of the AKR@ZIF-8 complex powder prepared in step 1) and 5 mL of crude FDH enzyme solution to the solution to obtain a mixed solution. Stir the mixed solution magnetically for 10 minutes to obtain a milky white viscous hybrid hydrogel precursor solution.

[0075] 3) Cross-linking and molding of AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres: Prepare a 100 mL aqueous solution containing 3% (w / v, g / mL) calcium chloride and 0.5% (w / v, g / mL) glutaraldehyde as a crosslinking solution. Using a 10 mL syringe with a 22G needle, add the entire hybrid hydrogel precursor solution prepared in step 2) dropwise at a flow rate of approximately 1 mL / min to the stirring crosslinking solution. After the addition is complete, continue stirring for 30 minutes and let stand for 2 hours to ensure complete crosslinking. Then filter and collect the hybrid hydrogel microspheres, and rinse several times with deionized water to remove residual calcium chloride and unreacted glutaraldehyde from the surface of the hybrid hydrogel microspheres. The resulting AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres are stored at 4℃ for later use.

[0076] Example 2 A method for preparing AKR@ZIF-8-FDH@SA / CS hybrid hydrogel microspheres, the specific preparation steps are as follows: 1) Preparation of AKR@ZIF-8 complex: Accurately weigh 0.4g of bacterial sludge containing aldehyde reductase, disperse it in 20mL of phosphate buffer solution (pH=7.0), sonicate to disrupt, centrifuge at 10000rpm for 5 minutes, and take the supernatant to obtain crude AKR enzyme solution (protein concentration: 4mg / mL). Weigh 2.27 g of anhydrous zinc acetate and dissolve it in 40 mL of deionized water to obtain a metal salt solution (0.31 M). Weigh 4.11 g of 2-methylimidazole and dissolve it in 400 mL of deionized water to obtain an organic ligand solution (1.25 M). Under room temperature and magnetic stirring, 20 mL of crude AKR enzyme solution and 400 mL of organic ligand solution were mixed evenly to obtain a mixed solution. 40 mL of metal salt solution was quickly added to the mixed solution, and the reaction was continued to be stirred for 30 min. After the reaction was completed, the product was centrifuged at 10000 rpm for 5 min and washed 3 times with deionized water. Finally, the product was freeze-dried to obtain AKR@ZIF-8 composite powder.

[0077] 2) Preparation of hybrid hydrogel precursor solution: Weigh 2g of bacterial sludge containing formate dehydrogenase (FDH), disperse it in 50mL of phosphate buffer solution (pH=7.0), sonicate to break it up, centrifuge at 10000rpm for 5 minutes, and take the supernatant to obtain crude FDH enzyme solution (protein concentration: 8mg / mL).

[0078] Weigh 0.5g of sodium alginate and add it to 25mL of deionized water. Heat and stir in a 60℃ water bath until completely dissolved. Then add 0.4g of chitosan and acetic acid to promote the dissolution of chitosan. After the chitosan is completely dissolved, adjust the pH of the solution to 6.5 with 0.5M sodium hydroxide solution and then cool to room temperature. Then, add 100mg of the AKR@ZIF-8 complex powder prepared in step 1) and 5mL of crude FDH enzyme solution to the solution to obtain a mixed solution. Stir the mixed solution magnetically for 10 minutes to obtain a milky white viscous hybrid hydrogel precursor solution.

[0079] 3) Cross-linking and molding of AKR@ZIF-8-FDH@SA / CS hybrid hydrogel microspheres Prepare a 100 mL crosslinking solution containing 2% (w / v, g / mL) calcium chloride and 1% (w / v, g / mL) glutaraldehyde. Using a 10 mL syringe with a 22G needle, aspirate the entire hybrid hydrogel precursor solution prepared in step 2) dropwise into the stirred mixture at a flow rate of approximately 1 mL / min. After the addition is complete, continue stirring for 30 minutes and let stand for 2 hours to ensure complete crosslinking. Then, filter and collect the hybrid microspheres, and rinse several times with deionized water to remove residual calcium chloride and unreacted glutaraldehyde from the surface of the hybrid microspheres. The resulting AKR@ZIF-8-FDH@SA / CS hybrid hydrogel microspheres are stored at 4°C for later use.

[0080] Example 3 A method for preparing GOx@ZIF-90-HRP@SA / gelatin, the specific preparation steps are as follows: 1) Preparation of GOx@ZIF-90 complex Weigh 50 mg of glucose oxidase (GOx) and dissolve it in 5 mL of deionized water to obtain a glucose oxidase solution; Dissolve 480 mg of 2-imidazolium formaldehyde and 50 mg of polyvinylpyrrolidone (PVP) in 20 mL of deionized water to obtain an organic ligand solution; Dissolve 371 mg of zinc nitrate hexahydrate in 25 mL of tert-butanol to obtain a metal salt solution; Under room temperature and magnetic stirring, 5 mL of glucose oxidase solution and 20 mL of organic ligand solution were mixed evenly to obtain a mixed solution. 25 mL of metal salt solution was quickly added to the mixed solution, and the reaction was continued to be stirred for 30 min. After the reaction was completed, the product was centrifuged at 10000 rpm for 5 min and washed 3 times with deionized water. Finally, the product was freeze-dried to obtain GOx@ZIF-90 complex powder.

[0081] 2) Preparation of hybrid hydrogel precursor solution: Weigh 0.5g sodium alginate and 10g gelatin and add them to 50mL deionized water. Heat and stir in a 60℃ water bath until completely dissolved, then cool to room temperature. Then, add 100mg of the GOx@ZIF-90 complex powder prepared in step 1) and 10mg of horseradish peroxidase (HRP) to the solution to obtain a mixed solution. Stir the mixed solution magnetically for 10 minutes to obtain a milky white viscous hybrid hydrogel precursor solution.

[0082] 3) Cross-linking molding of GOx@ZIF-90-HRP@SA / gelatin hybrid microspheres Prepare a 200 mL aqueous solution containing 3% (w / v, g / mL) calcium chloride and 2% (w / v, g / mL) glutaraldehyde as a crosslinking solution. Using a 10 mL syringe with a 22G needle, aspirate the entire hybrid hydrogel precursor solution prepared in step 2) in batches and add it dropwise at a flow rate of approximately 1 mL / min to the stirring crosslinking solution. After the addition is complete, continue stirring for 30 minutes and let it stand for 2 hours to ensure complete crosslinking. Then, filter and collect the hybrid hydrogel beads, and rinse several times with deionized water to remove residual calcium chloride and unreacted glutaraldehyde from the surface of the hybrid hydrogel beads. The resulting GOx@ZIF-90-HRP@SA / gelatin hybrid hydrogel beads are stored at 4°C for later use.

[0083] Example 1 of microbead catalytic reaction A method for the catalytic production of (R)-6-hydroxy-8-chlorooctanoate ethyl ester using AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads, the reaction equation of which is as follows: Figure 2 As shown, the specific steps include the following: 2 g of ethyl 6-carbonyl-8-chlorooctanoate was dissolved in 1 mL of methyl tert-butyl ether and then mixed with 19 mL of phosphate buffer (pH=7.0, 0.1M) to obtain a mixed solution. 1.42 g of ammonium formate and 4 mg of nicotinamide adenine dinucleotide phosphate (NADP+) were added to the above mixed solution and stirred until homogeneous. Then, the AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads prepared in Example 1 were added, and the reaction was carried out at 30 °C and 200 rpm for 4 hours.

[0084] Example 1 of glucose biosensor The application of GOx@ZIF-90-HRP@SA / gelatin hybrid hydrogel microbeads in a glucose biosensor is illustrated in the following method: 1) Thirty GOx@ZIF-90-HRP@SA / gelatin hybrid hydrogel microbeads prepared in Example 3 were placed in phosphate buffer solutions containing 10 mL of 10 mM 3,3',5,5'-tetramethylbenzidine (TMB) and different concentrations of glucose; the glucose concentrations were 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mM, respectively. In the GOx@ZIF-90-HRP@SA / gelatin hybrid hydrogel microbeads, GOx catalyzes the production of H2O2 from glucose. H2O2 is then utilized by HRP to catalyze the oxidation of TMB to a blue substrate. After 100 minutes of catalysis, the absorbance of solutions with different glucose concentrations at 650 nm (the wavelength of maximum absorbance of the blue product) was measured. A standard curve of absorbance versus glucose concentration was plotted. The standard curve is shown below. Figure 3 As shown; 2) Add 30 GOx@ZIF-90-HRP@SA / gelatin hybrid hydrogel microbeads prepared in Example 3 and 10 mM of 3,3',5,5'-tetramethylbenzidine (TMB) to the glucose phosphate buffer solution to be tested, and carry out the catalytic reaction. After the reaction is completed for 100 minutes, the absorbance value of the solution at 650 nm is measured, and the concentration of glucose in the glucose phosphate buffer solution to be tested is quantified according to the absorbance value-glucose concentration standard curve.

[0085] Comparative Example 1 A method for preparing ZIF-8 with co-immobilized AKR and FDH, the specific steps of which are as follows: 1) Accurately weigh 2g of bacterial sludge containing aldehyde reductase (AKR), disperse it in 100 mL of phosphate buffer solution (pH=7.0), sonicate to disrupt, centrifuge at 10000 rpm for 5 minutes, and collect the supernatant to obtain crude AKR enzyme solution (protein concentration: 4 mg / mL); weigh 2g of bacterial sludge containing formate dehydrogenase (FDH), disperse it in 100 mL of phosphate buffer solution (pH=7.0), sonicate to disrupt, centrifuge at 10000 rpm for 5 minutes, and collect the supernatant to obtain crude FDH enzyme solution (protein concentration: 4 mg / mL). 2) Dissolve 367 mg of anhydrous zinc acetate in 100 mL of deionized water to obtain a metal salt solution; weigh 657 mg of 2-methylimidazole and dissolve it in 100 mL of deionized water to obtain an organic ligand solution; mix 20 mL of crude AKR enzyme solution, 20 mL of crude FDH enzyme solution, and 100 mL of organic ligand solution evenly at room temperature and with magnetic stirring, then quickly add 100 mL of the metal salt solution. Continue stirring for 30 min. After the reaction is complete, centrifuge the product at 10000 rpm for 5 min, wash it three times with deionized water, and finally freeze-dry the product to obtain AKR-FDH@ZIF-8 complex powder.

[0086] Comparative Example 2 A method for preparing an SA / PVA mixed hydrogel with co-immobilized AKR and FDH, the specific steps of which are as follows: 1) Preparation of crude enzyme solution: Accurately weigh 2g of bacterial sludge containing aldehyde-ketone reductase (AKR), disperse it in 100 mL of phosphate buffer solution (pH=7.0), sonicate to disrupt the sludge, centrifuge at 10000 rpm for 5 minutes, and collect the supernatant to obtain crude AKR enzyme solution (protein concentration: 4 mg / mL); weigh 2g of bacterial sludge containing formate dehydrogenase (FDH), disperse it in 100 mL of phosphate buffer solution (pH=7.0), sonicate to disrupt the sludge, centrifuge at 10000 rpm for 5 minutes, and collect the supernatant to obtain crude FDH enzyme solution (protein concentration: 4 mg / mL). 2) Preparation of hybrid hydrogel precursor solution: Weigh 0.2 g sodium alginate (SA) and 0.2 g polyvinyl alcohol (PVA), add them to 10 mL of deionized water, heat and stir in a 60°C water bath until completely dissolved, and cool to room temperature; then add 5 mL of AKR crude enzyme solution and 5 mL of FDH crude enzyme solution prepared in step 1) to the solution, and magnetically stir the mixed solution for 10 minutes to obtain a transparent, viscous hydrogel precursor solution; 3) Cross-linking and molding of AKR-FDH@SA / PVA hybrid hydrogel microspheres: Prepare a 100 mL aqueous solution containing 3% (w / v) calcium chloride and 0.5% (w / v) glutaraldehyde as a crosslinking solution. Using a 10 mL syringe with a 22G needle, aspirate the entire hydrogel precursor solution prepared in step 2) in batches and add it dropwise at a flow rate of approximately 1 mL / min to the crosslinking solution while it is being stirred. After the addition is complete, continue stirring for 30 minutes and let it stand for 2 hours to ensure complete crosslinking. Then, filter and collect the microbeads, and rinse them several times with deionized water to remove residual calcium chloride and unreacted glutaraldehyde from the surface of the microbeads, obtaining AKR-FDH@SA / PVA hybrid hydrogel microbeads.

[0087] Catalytic reaction comparative example 1 The production of ethyl (R)-6-hydroxy-8-chlorooctanoate using the AKR-FDH@ZIF-8 complex obtained in Comparative Example 1 includes the following steps: Weigh 2g of ethyl 6-carbonyl-8-chlorooctanoate and dissolve it in 1mL of methyl tert-butyl ether. Then mix it with 19mL of phosphate buffer (pH=7.0, 0.1M) to obtain a mixed solution. Weigh 1.42g of ammonium formate and 4mg of nicotinamide adenine dinucleotide phosphate (NADP+) and add them to the above mixed solution. After stirring evenly, add the AKR-FDH@ZIF-8 complex prepared in Comparative Example 1. React in a shaker at 30℃ and 200 rpm for 4 hours until the reaction is complete.

[0088] Comparative Example 2 of Catalytic Reaction The production of (R)-6-hydroxy-8-chlorooctanoate ethyl catalysis using AKR-FDH@SA / PVA hybrid hydrogel microbeads obtained in Comparative Example 2 includes the following steps: 2 g of ethyl 6-carbonyl-8-chlorooctanoate was dissolved in 1 mL of methyl tert-butyl ether and then mixed with 19 mL of phosphate buffer (pH=7.0, 0.1M) to obtain a mixed solution. 1.42 g of ammonium formate and 4 mg of nicotinamide adenine dinucleotide phosphate (NADP+) were added to the above mixed solution and stirred until homogeneous. Then, the AKR-FDH@SA / PVA hybrid hydrogel microbeads prepared in Comparative Example 2 were added, and the reaction was carried out at 30 °C and 200 rpm for 4 hours. The reaction was then completed.

[0089] I. Morphology and Structural Characterization 1. Transmission electron micrographs and optical photographs: Figure 4 This is a transmission electron microscope (TEM) image of the AKR@ZIF-8 complex obtained in step 1) of Example 1. Figure 4 It can be seen that the prepared AKR@ZIF-8 complex has a regular morphology and uniform particle size.

[0090] Figure 5 Optical photograph of the AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres prepared in Example 1. Figure 5 It can be seen that the AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres prepared in Example 1 have a uniform particle size of about 1.5 mm.

[0091] Figure 6 Transmission electron microscopy (TEM) image of the AKR-FDH@ZIF-8 composite prepared in Comparative Example 1. Figure 6 It can be seen that the AKR-FDH@ZIF-8 complex prepared in Comparative Example 1 also has a regular morphology and uniform particle size.

[0092] Figure 7 Optical photograph of the AKR-FDH@SA / PVA hybrid hydrogel microspheres prepared for Comparative Example 2. Figure 7It can be seen that the AKR-FDH@SA / PVA hybrid hydrogel microspheres prepared in Comparative Example 2 have a uniform particle size of approximately 2.5 mm.

[0093] Figure 8 This is a transmission electron microscope (TEM) image of the GOx@ZIF-90 composite obtained in step 1) of Example 3. Figure 8 It can be seen that the GOx@ZIF-90 composite prepared in Example 3 has a regular morphology and uniform particle size.

[0094] Figure 9 Optical photograph of the GOx@ZIF-90-HRP@SA / gelatin hybrid hydrogel microspheres prepared in Example 3. Figure 9 It can be seen that the GOx@ZIF-90-HRP@SA / gelatin hybrid hydrogel microspheres prepared in Example 3 have a uniform particle size of about 2 mm.

[0095] 2. Structural characterization Figure 10 X-ray powder diffraction patterns of the AKR@ZIF-8 composite and AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres prepared in Example 1. Figure 10 In the middle (a), AKR@ZIF-8 is used. Figure 10 (b) shows AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres. (The image shows the microspheres formed by...) Figure 10 It can be seen that Example 1 successfully prepared the AKR@ZIF-8 complex, and successfully mixed and hybridized AKR@ZIF-8 in the AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres.

[0096] Figure 11 Fourier transform infrared spectra of the AKR@ZIF-8 complex obtained in step 1) of Example 1, the prepared AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres, the AKR-FDH@ZIF-8 complex prepared in Comparative Example 1, and the AKR-FDH@SA / PVA hybrid hydrogel microspheres prepared in Comparative Example 2. Figure 11 In the images, (a) represents the AKR@ZIF-8 complex, (b) represents the AKR-FDH@ZIF-8 complex, (c) represents AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres, and (d) represents AKR-FDH@SA / PVA hybrid hydrogel microspheres. Infrared spectroscopy results for AKR@ZIF-8 and AKR-FDH@ZIF-8 show that at 1600 cm⁻¹... -1The appearance of characteristic peaks of the protein indicates that ZIF-8 has successfully encapsulated the protease; the results of AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads show that the protease, ZIF-8 and polymers SA and PVA have been successfully complexed.

[0097] Figure 12 X-ray powder diffraction patterns of the GOx@ZIF-90 composite and GOx@ZIF-90-HRP@SA / gelatin hybrid hydrogel microspheres prepared in Example 3. Figure 12 It can be seen that Example 3 successfully prepared the GOx@ZIF-90 complex, and successfully mixed and hybridized GOx@ZIF-90 in the GOx@ZIF-90-HRP@SA / gelatin hybrid hydrogel microspheres.

[0098] II. Performance testing of catalytic conversion of ethyl 6-carbonyl-8-chlorooctanoate to (R)-6-hydroxy-8-chlorooctanoate In the reaction processes of Microbead Catalysis Example 1, Comparative Catalysis Example 1, and Comparative Catalysis Example 2, every hour, 200 μL of the reaction solution was taken, extracted with 200 μL of methyl tert-butyl ether, centrifuged, and the upper organic phase was collected for TLC testing. The developing solvent was petroleum ether:ethyl acetate 7:3 (v:v). The TLC test results are as follows. Figure 13 As shown. Figure 13 In the table, A1, A2, A3, and A4 represent the TLC detection results of the microbead catalytic reaction Example 1 at 1h, 2h, 3h, and 4h of catalytic conversion, respectively; B1, B2, B3, and B4 represent the TLC detection results of the catalytic reaction Comparative Example 1 at 1h, 2h, 3h, and 4h of catalytic conversion, respectively; and C1, C2, C3, and C4 represent the TLC detection results of the catalytic reaction Comparative Example 2 at 1h, 2h, 3h, and 4h of catalytic conversion.

[0099] Depend on Figure 13 It was observed that after 4 hours of reaction, both Comparative Examples 1 and 2 of the catalytic reaction could only catalyze the conversion of approximately 20% of ethyl 6-carbonyl-8-chlorooctanoate. However, in Example 1 of the microbead catalytic reaction, the catalytic conversion rate of ethyl 6-carbonyl-8-chlorooctanoate reached over 90% after 3 hours, and the substrate was almost invisible after 4 hours, indicating that almost all of it was converted into product. This demonstrates that, based on the layered co-immobilization of two enzymes proposed in this invention, the AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads prepared in Example 1 of this invention effectively isolate the AKR and FDH enzymes, avoiding direct contact and potential mutual inhibition between the two enzymes, while also shortening the diffusion distance of intermediate products, thus greatly improving the overall efficiency of the cascade reaction.

[0100] In Example 1 of the microbead-catalyzed reaction, after a 4-hour reaction, 200 μL of the reaction solution was taken, extracted with 200 μL of methyl tert-butyl ether, centrifuged, and the upper organic phase was collected. Gas chromatography was used to determine the concentrations and chirality of ethyl 6-carbonyl-8-chlorooctanoate and (R)-6-hydroxy-8-chlorooctanoate. The detection results are as follows: Figure 14 and Figure 15 As shown.

[0101] Figure 14 The gas chromatogram for the conversion of ethyl 6-carbonyl-8-chlorooctanoate to (R)-6-hydroxy-8-chlorooctanoate after 4 hours of catalytic reaction in Example 1 of the microbead catalysis reaction is shown in Table 1. The gas chromatographic peaks of the substrate (ethyl 6-carbonyl-8-chlorooctanoate) and the product (ethyl 6-hydroxy-8-chlorooctanoate) are shown in Table 1.

[0102] Table 1 Gas chromatographic peaks of substrates and products

[0103] Depend on Figure 14 As shown in Table 1, after 4 hours of reaction, the catalytic conversion rate of 6-carbonyl-8-chlorooctanoate by the AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres prepared in Example 1 reached over 97.5%.

[0104] Figure 15 The chiral test results of (R)-6-hydroxy-8-chlorooctanoate ethyl ester prepared by the microbead catalysis in Example 1 are shown in Table 2. The gas chromatographic peak tables of (R)-6-hydroxy-8-chlorooctanoate ethyl ester and (S)-6-hydroxy-8-chlorooctanoate ethyl ester are also shown in Table 2.

[0105] Table 2 Gas chromatographic peaks of (R)-6-hydroxy-8-chlorooctanoate ethyl ester and (S)-6-hydroxy-8-chlorooctanoate ethyl ester

[0106] Depend on Figure 15 As shown in Table 2, the ethyl (R)-6-hydroxy-8-chlorooctanoate catalyzed by the AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads prepared in Example 1 has an ee value greater than 99%, indicating high purity of the catalytic product.

[0107] III. Recyclability Test The recyclability and stability of the AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres prepared in Example 1 were tested. Specifically, following the catalytic method of Example 1 for microsphere catalysis, after 4 hours of reaction, the microspheres were filtered and recovered, repeatedly washed with phosphate buffer (pH=7.0, 0.1 M), and then soaked in 50 mL of 3% (w / v) calcium chloride solution for 2 hours. The microspheres were then removed and the reaction was continued according to the catalytic reaction method of Example 1 for microsphere catalysis. This procedure was repeated for 5 cycles. After each reaction, the filtrate was collected and extracted with an equal volume of methyl tert-butyl ether. The upper organic phase was collected, dehydrated with anhydrous sodium sulfate, and then rotary evaporated to obtain the product. The product was analyzed by gas chromatography, and the product conversion rate was calculated based on the peak area. The product conversion rate of the first cycle was taken as 100%, and the ratio of the product conversion rate of each subsequent cycle to the product conversion rate of the first cycle was the relative enzyme activity.

[0108] Figure 16 The relative enzyme activity was measured after five cycles of catalysis using AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel beads. Figure 16 It can be seen that after 5 cycles of the reaction, the AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads still maintained more than 85% of the enzyme activity, which shows that the AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads of the present invention have excellent recyclability.

[0109] IV. Storage Stability Test 1. The AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres and free AKR prepared in Example 1 were stored at 30°C for 7 days, and samples were taken periodically to test their activity.

[0110] Activity assay method: In a 1cm quartz cuvette, add 2mL of preheated (30℃) pH 7.0, 0.1M phosphate buffer solution, 10μL of 0.048M reduced nicotinamide adenine dinucleotide phosphate (NADPH) solution, and 10μL of ethyl 6-carbonyl-8-chlorooctanoate. Mix well, then add 10μL of free AKR enzyme solution (or an equal amount of AKR enzyme AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel beads), shake quickly, and record the activity every 15 seconds for 120 seconds. 340 Absorbance change value. The enzyme activity is calculated based on the amount of enzyme required to consume 1 μmol of NADPH per minute, as shown in Formula I:

[0111] Formula I in, A represents the change in absorbance of NADPH at 340 nm over 60 seconds; V represents the reaction volume (2.0 mL); T represents the reaction time (60 s); m represents the amount of enzyme added; b represents the molar extinction coefficient of NADPH, which is 6220 L / (mol). (cm), where l is the optical path length of the cuvette: 1cm. The initial enzyme activity is taken as 100%, and the ratio of subsequent enzyme activities to this initial enzyme activity is the relative enzyme activity.

[0112] Figure 17 The figure shows the storage stability test results of free AKR and AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads at 30°C. Figure 17 It can be seen that the relative enzyme activity of free AKR is only 20% after 140 hours of storage; while the relative enzyme activity of AKR immobilized by AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads is still more than 50% after 168 hours of storage, indicating that AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads significantly improve the storage stability of AKR. 2. The AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microspheres and free FDH prepared in Example 1 were stored at 30°C for 7 days, and samples were taken periodically to test their activity.

[0113] Activity assay method: In a 1cm quartz cuvette, add 1.75 mL of preheated (30℃) pH 7.0, 0.1M phosphate buffer solution, 100 μL of 4M ammonium formate solution, and 100 μL of 30mM DADP+ solution. Mix well, then add 50 μL of free FDH enzyme solution (or an equal amount of FDH enzyme AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel beads). Shake quickly and record A values ​​at 15-second intervals over 120 seconds. 340 Absorbance change value. The enzyme activity is determined by the amount of enzyme required to generate 1 μmol NADPH per minute, calculated using Formula I. The initial enzyme activity is taken as 100%, and the enzyme activity in subsequent tests relative to this value is the relative enzyme activity.

[0114] Figure 18 The figure shows the storage stability test results of FDH immobilized in free FDH and AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads at 30°C. Figure 18 It can be seen that the relative enzyme activity of free FDH is only 10% after 96 hours of storage; while the relative enzyme activity of FDH immobilized by AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads is still more than 40% after 168 hours of storage. This indicates that AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads significantly improve the storage stability of FDH.

[0115] In summary, this invention employs a layered strategy of encapsulating the first enzyme within a MOF to prepare a first enzyme@MOF complex, and immobilizing the second enzyme and the first enzyme@MOF complex within a hydrogel. This effectively isolates the two enzymes, avoiding direct contact and potential mutual inhibition, while simultaneously shortening the diffusion distance of intermediate products and significantly improving the overall efficiency of the cascade reaction. Using the AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads prepared according to this invention to catalyze the production of ethyl (R)-6-hydroxy-8-chlorooctanoate, the catalytic conversion efficiency reached over 97.5% after 4 hours of catalytic reaction, while single MOF-immobilized enzymes and single hydrogel-immobilized enzymes could only catalyze the conversion of approximately 20% of ethyl 6-carbonyl-8-chlorooctanoate. Furthermore, the ethyl (R)-6-hydroxy-8-chlorooctanoate obtained by the AKR@ZIF-8-FDH@SA / PVA hybrid hydrogel microbeads of this invention has an ee value greater than 99%, indicating high purity of the catalytic product.

[0116] Compared to free enzymes, the MOF hybrid hydrogel microbeads with immobilized dual enzymes of this invention maintained more than 85% of their enzyme activity after 5 cycles of cycling. After 168 hours of storage, the MOF hybrid hydrogel microbeads with immobilized dual enzymes of this invention still had more than 50% of their relative enzyme activity. The MOF hybrid hydrogel microbeads with immobilized dual enzymes of this invention are far superior to free enzymes in terms of thermal stability, operational stability, long-term storage stability, and recyclability, and have good applications in the fields of constructing biosensors, producing pharmaceutical intermediates, synthesizing fine chemicals, and degrading environmental pollutants.

[0117] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A MOF hybrid hydrogel microsphere with immobilized dual enzymes, characterized in that, The dual enzymes are a first enzyme and a second enzyme that constitute a cascade catalytic reaction. The MOF hybrid hydrogel microbeads with the immobilized dual enzymes have a spatial layered structure, which includes an MOF that encapsulates the first enzyme and a hydrogel loaded with the second enzyme distributed around the MOF.

2. The MOF hybrid hydrogel microbeads with immobilized dual enzymes according to claim 1, characterized in that, The particle size of the MOF hybrid hydrogel microbeads with immobilized dual enzymes is 1~3 mm.

3. The MOF hybrid hydrogel microbeads with immobilized dual enzymes according to claim 1, characterized in that, The first enzyme is selected from one of aldehyde-ketone reductase, alcohol dehydrogenase, glucose oxidase, transaminase, amylase, peroxidase, pyruvate dehydrogenase, pectinase, carbon dioxide dehydrogenase, monooxygenase, urease, cholinesterase, and lipase; and / or, the second enzyme is selected from one of formate dehydrogenase, catalase, horseradish peroxidase, glutamate dehydrogenase, glucoamylase, laccase, lactate dehydrogenase, cellulase, formate dehydrogenase, glucose dehydrogenase, peroxidase, and esterase.

4. The method for preparing MOF hybrid hydrogel microbeads with immobilized dual enzymes as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1: Encapsulate the first enzyme within the MOF to prepare the first enzyme@MOF complex; S2: The first enzyme@MOF complex, the second enzyme, and the hydrogel components are dispersed in the medium to form a hybrid hydrogel precursor solution; S3: The hybrid hydrogel precursor solution is reacted with a crosslinking agent to form MOF hybrid hydrogel microbeads with immobilized dual enzymes.

5. The method for preparing MOF hybrid hydrogel microspheres with immobilized dual enzymes according to claim 4, characterized in that, The MOF is at least one of ZIF-8, ZIF-90, ZPF-2, ZIF-67, and ZIF-L.

6. The method for preparing MOF hybrid hydrogel microspheres with immobilized dual enzymes according to claim 4, characterized in that, The method for encapsulating the first enzyme within the MOF in S1 involves adding a metal salt solution of the MOF to a mixed solution of the organic ligand solution of the MOF and the first enzyme for in-situ self-assembly reaction. After the reaction is complete, centrifugation is performed to obtain the first enzyme@MOF complex.

7. The method for preparing MOF hybrid hydrogel microspheres with immobilized dual enzymes according to claim 6, characterized in that, The molar ratio of the metal salt of the MOF to the organic ligand of the MOF is (1:4) to (1:40).

8. The method for preparing MOF hybrid hydrogel microbeads with immobilized dual enzymes according to claim 4, characterized in that, The hydrogel component includes alginate and auxiliary polymers; the auxiliary polymers include at least one of chitosan, gelatin, starch, carboxymethyl cellulose, hydroxypropyl methyl cellulose, pectin, polyvinyl alcohol, polyethylene glycol, polyacrylamide, and polylactic acid.

9. The method for preparing MOF hybrid hydrogel microspheres with immobilized dual enzymes according to claim 4, characterized in that, The crosslinking agent in S3 includes metal ion crosslinking agents and / or chemical crosslinking agents; the metal ions in the metal ion crosslinking agent include Ca... 2+ Zn 2+ Mg 2+ Al 3+ Fe 2+ Fe 3+ At least one of the following; the chemical crosslinking agent includes at least one of glutaraldehyde, acrolein, and glyoxal.

10. The application of the MOF hybrid hydrogel microspheres with immobilized dual enzymes as described in any one of claims 1 to 3 in the fields of constructing biosensors, producing pharmaceutical intermediates, synthesizing fine chemicals, and degrading environmental pollutants.

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