Preparation method of covalent organic framework microcapsule immobilized enzyme and application thereof

Abstract

This invention discloses a method for preparing covalent organic framework microcapsules immobilized enzymes and their applications. Using sodium polystyrene sulfonate-doped calcium carbonate as a template, polyethyleneimine and enzyme molecules are sequentially adsorbed onto the calcium carbonate surface based on electrostatic interactions. Trialdehyde phloroglucinol and benzidine are simultaneously added to the calcium carbonate template with the adsorbed enzyme. Based on the enzyme-induced catalytic effect, covalent organic framework microcapsules are generated on the calcium carbonate surface. After post-treatment to remove the calcium carbonate template, the covalent organic framework microcapsule immobilized enzyme is finally obtained. When used for the conversion of pyruvate to lactate, the immobilized enzyme activity retains 70% of the free enzyme activity. After eight cycles, the immobilized enzyme activity still retains 89% of the initial activity, exhibiting high immobilized enzyme activity and stability. The preparation method is simple, environmentally friendly, and has significant application potential.

Description

Technical Field

[0001] This invention relates to a method for preparing microencapsulated immobilized enzymes and their applications, particularly a method for preparing microencapsulated immobilized enzymes for catalyzing the conversion of pyruvate to lactate and their applications. Background Technology

[0002] Enzymes are a core component of synthetic biology and green biomanufacturing, and immobilization offers a simple and feasible strategy to facilitate the industrialization of enzymes in pharmaceuticals, biosensors, and biosynthesis. Over the past few decades, researchers have focused on exploring the use of polymers, inorganic materials, and hybrid materials for synthesizing immobilization supports. However, low enzyme activity remains a challenge for current immobilization technologies. Therefore, synthesizing an immobilization support that maintains both high enzyme activity and high stability is crucial. As the core component of enzyme immobilization, the support material should possess characteristics such as good stability, suitable pore structure, and ease of surface modification.

[0003] Based on this, the present invention provides a method for preparing covalent organic framework (COF) microencapsulated enzymes. Utilizing the excellent structural stability, high surface area, and pore regularity of COFs, the resulting microcapsule structure provides a broad microenvironment for the enzyme, resulting in enhanced stability and reduced enzyme leakage. In addition to the capsule cavity, the capsule wall can also effectively immobilize the enzyme. Immobilizing the enzyme on the capsule wall fully utilizes the structural advantages of COF materials, shortening the mass transfer distance, reducing diffusion resistance, and ultimately achieving high immobilized enzyme activity and stability. In this invention, covalent organic framework microcapsules are constructed on the surface of calcium carbonate microspheres using the electrostatic interactions between sodium polystyrene sulfonate, polyethyleneimine, and enzyme molecules. The immobilized enzyme is obtained by removing the calcium carbonate using an etching method. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing covalently encapsulated enzymes. Compared with in-situ encapsulation of covalently encapsulated enzymes, the method provided by this invention significantly improves enzyme activity and cycling stability by immobilizing the enzyme on the capsule wall.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention proposes a method for preparing covalently organic framework microcapsules immobilized enzymes. The main steps are as follows: using sodium polystyrene sulfonate-doped calcium carbonate as a template, polyethyleneimine and an enzyme solution are added sequentially. The enzyme is adsorbed onto the calcium carbonate template surface using electrostatic interactions. Trialdehyde-based phloroglucinol and benzidine are simultaneously added to the enzyme-adsorbed calcium carbonate template. The enzyme-induced catalytic effect on the trialdehyde-based phloroglucinol and benzidine generates covalently organic framework microcapsules on the calcium carbonate template surface. After post-treatment to remove the calcium carbonate template, the covalently organic framework microcapsules immobilized enzyme are finally obtained. The specific steps are as follows:

[0007] Step 1: Preparation of sodium polystyrene sulfonate-doped calcium carbonate template; Prepare a 0.33 mol / L calcium chloride solution, add sodium polystyrene sulfonate to it to a final concentration of 3.0 mg / mL, and denote the above solution as solution A; after magnetic stirring until homogeneous, quickly add a sodium carbonate solution with the same molar concentration and volume as the calcium chloride solution to solution A, stir at 1200 r / min for 30 s, and let stand. The resulting precipitate is the sodium polystyrene sulfonate-doped calcium carbonate template (abbreviated as P@CaCO3).

[0008] Step 2: Preparation of calcium carbonate template for surface adsorption of enzyme; Polyethylene imine is added to the P@CaCO3 solution obtained in Step 1 to obtain solution B. After stirring evenly, the supernatant is removed, and the solution is centrifuged and washed with water. The resulting precipitate is the calcium carbonate template with polyethylene imine adsorbed on its surface (abbreviated as PP@CaCO3); The enzyme and polyvinylpyrrolidone are mixed at a mass ratio of 1:2, and then phosphate buffer is added, denoted as solution C; After complete dissolution, the above solution C is added to the PP@CaCO3 precipitate and stirred evenly to obtain the calcium carbonate template solution with enzyme adsorbed on its surface (abbreviated as PP@CaCO3@LDH), denoted as solution D;

[0009] Step 3: Preparation of calcium carbonate template for surface-formed covalent organic framework microcapsules; prepare ethanol solutions of trialdehyde resorcinol and benzidine at molar concentrations of 18.7 mmol / L and 28.2 mmol / L, respectively; simultaneously add the ethanol solutions of trialdehyde resorcinol and benzidine to the PP@CaCO3@LDH solution obtained in Step 2 to obtain solution E; in solution E, the molar concentration of trialdehyde resorcinol is 8.3 mmol / L and the molar concentration of benzidine is 12.5 mmol / L; stir the above solution E for 5-45 min, remove the supernatant, centrifuge and wash with water to obtain the calcium carbonate template for surface-formed covalent organic framework microcapsules (abbreviated as PP@CaCO3@LDH@TpBD);

[0010] Step 4: Preparation of covalent organic framework microencapsulated enzyme; The PP@CaCO3@LDH@TpBD obtained in Step 3 is post-treated to remove the calcium carbonate template, and then centrifuged and washed with water; Finally, the covalent organic framework microencapsulated enzyme is obtained, which is referred to as LDH@TpBD microcapsule in this invention.

[0011] Furthermore, in the method for preparing covalently organic framework microencapsulated enzymes according to the present invention, wherein:

[0012] In step two, the enzyme is lactate dehydrogenase, which catalyzes the conversion of pyruvate into lactate.

[0013] In step four, the post-processing method involves dispersing the calcium carbonate template with covalent organic framework microcapsules formed on the surface into an ethylenediaminetetraacetic acid solution.

[0014] Furthermore, this invention also proposes a covalent organic framework microcapsule immobilized enzyme obtained using the aforementioned preparation method.

[0015] The covalent organic framework microencapsulated enzyme has a particle size of 2.56-3.35 μm, with an atomic percentage of approximately 76.35% carbon, approximately 8.61% nitrogen, approximately 14.49% oxygen, and approximately 0.55% sulfur.

[0016] Application: The covalent organic framework microcapsule immobilized enzyme prepared according to this invention was used to catalyze the conversion of pyruvate to lactate. The reaction was carried out at 45°C for 15 min. The immobilized enzyme activity was calculated based on the reaction rate, and the immobilized enzyme activity was found to be 70% of the free enzyme activity. Moreover, after being recycled 8 times, the immobilized enzyme activity still reached 89% of the initial activity.

[0017] The advantages of the covalent organic framework microcapsule immobilized enzyme preparation method proposed in this invention are: mild preparation conditions, simple preparation process, and effective shortening of mass transfer distance and reduction of diffusion resistance by immobilizing the enzyme in the microcapsule wall, thereby improving enzyme activity. The size and morphology of the covalent organic framework microcapsules can be controlled by changing the rotation speed, concentration of trialdehyde phloroglucinol, concentration of benzidine, concentration of organic solvent, and reaction time during the preparation process, thus obtaining microcapsule immobilized enzymes with different activities. Compared with in-situ embedded covalent organic framework immobilized enzymes, the covalent organic framework microcapsule immobilized enzyme prepared in this invention exhibits an improved reaction rate catalyzed by the substrate, retains 70% of the activity of the free enzyme, and can be separated and recovered. After 8 cycles, it retains 89% of the initial immobilized enzyme activity, while the activity of the in-situ embedded covalent organic framework immobilized enzyme is only 75% of the initial immobilized enzyme activity after 8 cycles. Compared to in-situ encapsulated covalent organic frameworks, enzyme activity and cycling stability are significantly improved, demonstrating industrialization potential. Attached Figure Description

[0018] Figure 1 The image shows a scanning electron microscope (SEM) image of the in-situ embedded covalent organic framework particles prepared in Comparative Example 1.

[0019] Figure 2 Here is a scanning electron microscope image of the covalent organic framework microcapsules prepared in Example 1;

[0020] Figure 3 This is a comparison of the reaction rates of the covalent organic framework microcapsule-immobilized enzymes prepared in Comparative Example 1 and Example 1 during use.

[0021] Figure 4 A comparison diagram showing the activity of the immobilized enzymes in covalent organic framework microcapsules prepared in Example 1 and Example 1 during recycling.

[0022] Figure 5 Scanning electron microscope (SEM) images of the covalent organic framework microcapsules prepared in Examples 1, 2, 3, and 4;

[0023] Figure 6 The graph shows a comparison of the reaction rates of the covalent organic framework microcapsule-immobilized enzymes prepared in Examples 1, 5, 6, 7, 8, and 9 during their use. Detailed Implementation

[0024] This invention proposes a method for preparing covalent organic framework (COF) microencapsulated enzymes. The design concept is to immobilize the enzyme on the capsule wall. Microencapsulation of COFs can reduce mass transfer resistance, making the covalent organic framework microencapsulated enzymes more effective in maintaining enzyme activity and stability. The basic scheme involves: adsorbing the enzyme onto the surface of a calcium carbonate template using electrostatic interactions; simultaneously adding trialdehyde phloroglucinol and benzidine to the enzyme-adsorbed calcium carbonate template; and utilizing the enzyme's inductive catalytic effect on the trialdehyde phloroglucinol and benzidine to generate covalent organic framework microcapsules on the calcium carbonate template surface; after post-treatment to remove the calcium carbonate template, the covalent organic framework microencapsulated enzyme is finally obtained. The main steps include: preparing a calcium carbonate template doped with sodium polystyrene sulfonate; adding polyethyleneimine and an enzyme solution sequentially to the calcium carbonate template solution doped with sodium polystyrene sulfonate and shaking the mixture; centrifuging the resulting solution to remove the supernatant; washing the mixture with deionized water to obtain a calcium carbonate template with enzyme adsorption on its surface; adding trialdehyde phloroglucinol and benzidine to the enzyme-adsorbed calcium carbonate template solution and stirring the mixture; centrifuging the resulting solution to remove the supernatant; washing the mixture with deionized water to obtain a calcium carbonate template with covalent organic framework microcapsules on its surface; and post-processing to remove the calcium carbonate template to obtain enzyme immobilized in covalent organic framework microcapsules.

[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, comparative examples and specific embodiments. The specific embodiments described are only for explanation and illustration of the present invention and are not intended to limit the present invention.

[0026] Comparative Example 1

[0027] The following steps were taken to prepare an in-situ embedded covalent organic framework immobilized enzyme, referred to as LDH@TpBD particles:

[0028] Step 1: Mix 0.56 mg of enzyme and polyvinylpyrrolidone at a mass ratio of 1:2, then add 5 mL of phosphate buffer; stir the above solution until fully dissolved;

[0029] Step 2: Prepare equal volumes of ethanol solutions of trialdehyde resorcinol and benzidine at molar concentrations of 18.7 mmol / L and 28.2 mmol / L, respectively. Simultaneously add the ethanol solutions of trialdehyde resorcinol and benzidine to the polyvinylpyrrolidone enzyme solution obtained in Step 1, resulting in a final molar concentration of 8.3 mmol / L for trialdehyde resorcinol and 12.5 mmol / L for benzidine. Stir the solution for 5–45 min, remove the supernatant, centrifuge, and wash with water to obtain LDH@TpBD particles. After drying, each particle weighs approximately 10 mg, with an enzyme loading of 50 μg / mg. The particle size of LDH@TpBD particles ranges from 247.83 nm to 378.26 nm. The particles exhibit a spherical aggregated state. The catalytic reaction rate for the conversion of pyruvate to lactate is 51.91 μM min. -1 .

[0030] The LDH@TpBD particles obtained in Comparative Example 1 were added to the substrate pyruvate at 45 °C for catalytic reaction. The reaction rate was recorded for the first 15 min to calculate the immobilized enzyme activity. The content of the reactant NADH was detected by UV-Vis spectrophotometer.

[0031] Immobilized enzyme activity was calculated based on the concentration of substrate catalyzed by the enzyme per unit time. Calculations showed that the immobilized enzyme prepared in Comparative Example 1 had an activity that was 23% of that of the free enzyme. For example... Figure 3 As shown, after 8 cycles, the immobilized enzyme activity was 75% of the initial immobilized enzyme activity.

[0032] Example 1

[0033] The steps for preparing enzyme-immobilized enzymes in covalent organic framework microcapsules, abbreviated as LDH@TpBD microcapsules, are as follows:

[0034] Step 1: Preparation of sodium polystyrene sulfonate-doped calcium carbonate template; Prepare a 0.33 mol / L calcium chloride solution, add sodium polystyrene sulfonate to it to a final concentration of 3.0 mg / mL, and denote the above solution as solution A. After magnetic stirring until homogeneous, quickly add a sodium carbonate solution with the same molar concentration and volume as the calcium chloride solution to the above solution. Stir at 1200 r / min for 30 s and let stand. The resulting precipitate is sodium polystyrene sulfonate-doped calcium carbonate template (abbreviated as P@CaCO3), with a precipitate mass of approximately 0.2 g;

[0035] Step 2: Preparation of calcium carbonate template for surface adsorption of enzyme; Add 10 mg of polyethyleneimine to the P@CaCO3 solution obtained in Step 1, stir well, remove the supernatant, centrifuge and wash with water, and the resulting precipitate is the calcium carbonate template with polyethyleneimine adsorbed on the surface (abbreviated as PP@CaCO3); Mix the enzyme and polyvinylpyrrolidone at a mass ratio of 1:2, with a specific enzyme amount of 0.56 mg, then add 5 mL of phosphate buffer, dissolve thoroughly, and add to the PP@CaCO3 precipitate, stir well, and obtain the calcium carbonate template solution with enzyme adsorbed on the surface (abbreviated as PP@CaCO3@LDH).

[0036] Step 3: Preparation of calcium carbonate template for surface-formed covalent organic framework microcapsules; equal volumes of ethanol solutions of trialdehyde resorcinol and benzidine were prepared at molar concentrations of 18.7 mmol / L and 28.2 mmol / L, respectively; the ethanol solutions of trialdehyde resorcinol and benzidine were simultaneously added to the PP@CaCO3@LDH solution obtained in Step 2, resulting in a final molar concentration of 8.3 mmol / L for trialdehyde resorcinol and 12.5 mmol / L for benzidine; the solution was stirred for 30 min, the supernatant was removed, and the mixture was centrifuged and washed with water to obtain the calcium carbonate template for surface-formed covalent organic framework microcapsules (abbreviated as PP@CaCO3@LDH@TpBD);

[0037] Step 4: Preparation of enzyme immobilized in covalent organic framework microcapsules; The PP@CaCO3@LDH@TpBD obtained in Step 3 was post-treated to remove the calcium carbonate template, then centrifuged and washed with water; finally, LDH@TpBD microcapsules were obtained. After drying, the microcapsules weighed approximately 5 mg, with an enzyme loading of 85.9 μg / mg.

[0038] Elemental analysis was performed on the LDH@TpBD microcapsules prepared in Example 1. The atomic percentages of the elements were determined by X-ray photoelectron spectroscopy (XPS). The atomic percentages were: carbon 76.35%, nitrogen 8.61%, oxygen 14.49%, and sulfur 0.55%. A scanning electron microscope image of the LDH@TpBD microcapsules prepared in Example 1 is shown below. Figure 2 The LDH@TpBD microcapsules have a particle size of 2.56-3.35 μm. The microcapsule surface has a sheet-like stacked structure. Figure 6 The calculated reaction rate for the catalytic conversion of pyruvate to lactate was 158.0 μM min. -1 .

[0039] The LDH@TpBD microcapsules obtained in Example 1 were added to the substrate pyruvate at 45°C for catalytic reaction. The reaction rate was recorded for the first 15 minutes to calculate the immobilized enzyme activity. The content of the reactant NADH was detected by UV-Vis spectrophotometer.

[0040] Immobilized enzyme activity was calculated based on the concentration of substrate catalyzed by the enzyme per unit time. Calculations showed that the immobilized enzyme activity of the LDH@TpBD microcapsules prepared in Example 1 was 70% of that of the free enzyme, which is 3.04 times that of Comparative Example 1. Figure 3 As shown, after 8 cycles, the immobilized enzyme activity was 89% of the initial immobilized enzyme activity. Compared with the LDH@TpBD particles prepared in Comparative Example 1, the cycling stability of the LDH@TpBD microcapsules prepared in Example 1 was significantly improved.

[0041] Example 2

[0042] The preparation of LDH@TpBD-20 min microcapsules followed essentially the same steps as Example 1, except that in step three, after adding an ethanol solution of benzidine and trialdehyde-resorcinol, the reaction time was reduced from 30 min to 20 min. The final product obtained was LDH@TpBD-20 min microcapsules. After drying, the microcapsules weighed approximately 5 mg, with an enzyme loading of 83.1 μg / mg. The scanning electron microscope image is shown below. Figure 5 The particle size of the LDH@TpBD-20 min microcapsules was 2.55-3.38 μm. The reaction rate was 151.7 μM / min. -1 .

[0043] Example 3

[0044] The preparation of LDH@TpBD-10 min microcapsules followed essentially the same steps as Example 1, except that in step three, after adding an ethanol solution of benzidine and trialdehyde-resorcinol, the reaction time was reduced from 30 min to 10 min. The final product obtained was LDH@TpBD-10 min microcapsules. After drying, the microcapsules weighed approximately 3 mg, with an enzyme loading of 127.9 μg / mg. The scanning electron microscope image is shown below. Figure 5 The LDH@TpBD-10 min microcapsules have a particle size of 2.33-3.34 μm. Their catalytic reaction rate for the conversion of pyruvate to lactate is 101.3 μM min. -1 .

[0045] Example 4

[0046] The preparation of LDH@TpBD-5 min microcapsules followed essentially the same steps as Example 1, except that in step three, after adding an ethanol solution of benzidine and trialdehyde-resorcinol, the reaction time was reduced from 30 min to 5 min. The final product obtained was LDH@TpBD-5 min microcapsules. After drying, the microcapsules weighed approximately 2 mg, with an enzyme loading of 153.8 μg / mg. The scanning electron microscope image is shown below. Figure 5 The LDH@TpBD-5 min microcapsules have a particle size of 2.35-3.78 μm. Their catalytic reaction rate for the conversion of pyruvate to lactate is 46.4 μM min. -1 .

[0047] Example 5

[0048] Preparation of LDH 1.12 @TpBD microcapsules, Example 2 is basically the same as Example 1 in terms of steps, except that in step 2, the molecular weight of the added enzyme is changed from 0.56 mg to 1.12 mg, and the final product obtained is LDH. 1.12 @TpBD microcapsules. After drying, the microcapsules weighed approximately 5 mg, with an enzyme loading of 96.6 μg / mg.

[0049] Figure 6 The reaction rate for the catalytic conversion of pyruvate to lactate can be calculated to be 142.3 μM min. -1

[0050] Example 6

[0051] Preparation of LDH 0.28 @TpBD microcapsules, Example 2 is basically the same as Example 1 in terms of steps, except that in step 2, the molecular weight of the added enzyme is changed from 0.56 mg to 0.28 mg, and the final product obtained is LDH. 0.28 @TpBD microcapsules. After drying, the microcapsules weighed approximately 5 mg, with an enzyme loading of 37.5 μg / mg.

[0052] Figure 6 The reaction rate for the catalytic conversion of pyruvate to lactate can be calculated to be 78.8 μM min. -1

[0053] Example 7

[0054] Preparation of LDH 0.14 @TpBD microcapsules, Example 2 is basically the same as Example 1 in terms of steps, except that in step 2, the molecular weight of the added enzyme is changed from 0.56 mg to 0.14 mg, and the final product obtained is LDH.0.14 @TpBD microcapsules. After drying, the microcapsules weighed approximately 5 mg, with an enzyme loading of 22.0 μg / mg.

[0055] Figure 6 The reaction rate for the catalytic conversion of pyruvate to lactate can be calculated to be 48.2 μM min. -1

[0056] Example 8

[0057] Preparation of LDH 0.07 @TpBD microcapsules, Example 2 is basically the same as Example 1 in terms of steps, except that in step 2, the molecular weight of the added enzyme is changed from 0.56 mg to 0.07 mg, and the final product obtained is LDH. 0.07 @TpBD microcapsules. After drying, the microcapsules weighed approximately 5 mg, with an enzyme loading of 11.3 μg / mg.

[0058] Figure 6 The reaction rate for the catalytic conversion of pyruvate to lactate can be calculated to be 8.1 μM min. -1

[0059] In summary, by comparing the SEM images corresponding to Examples 1-4 with the immobilized enzyme reaction rate comparisons for Examples 1 and 5-7, it was found that using sodium polystyrene sulfonate-doped calcium carbonate as a template, and utilizing the enzyme's induced catalytic effect on the Schiff base reaction between trialdehyde resorcinol and benzidine, covalent organic framework microcapsules LDH@TpBD were ultimately obtained. Through time control, it was concluded that a more complete and stable LDH@TpBD microcapsule structure was obtained after a reaction time of 30 min. Figure 6 The results show that the best performance was achieved with the addition of 0.56 mg of enzyme. The covalently organic framework microcapsule-immobilized enzyme finally prepared in this invention retained 70% of the activity of the free enzyme, and after 8 cycles, it still retained 89% of the initial immobilized activity, exhibiting excellent reusability. When used to catalyze the conversion of pyruvate to lactate, it can significantly reduce costs.

[0060] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for preparing a covalently organic framework microencapsulated enzyme, characterized in that, Includes the following steps: Step 1: Preparation of sodium polystyrene sulfonate-doped calcium carbonate template: A 0.33 mol / L calcium chloride solution was prepared, and sodium polystyrene sulfonate with a final concentration of 3.0 mg / mL was added to it. This solution was denoted as solution A. After magnetic stirring until homogeneous, sodium carbonate solution with the same molar concentration and volume as the calcium chloride solution was quickly added to solution A. The solution was stirred at 1200 r / min for 30 s and then allowed to stand. The resulting precipitate was a sodium polystyrene sulfonate-doped calcium carbonate template P@CaCO3. Step 2: Preparation of calcium carbonate template for surface adsorption enzymes: Polyethyleneimine was added to the P@CaCO3 solution obtained in step one to obtain solution B; after stirring evenly, the supernatant was removed, and the solution was centrifuged and washed with water. The resulting precipitate was a calcium carbonate template PP@CaCO3 with polyethyleneimine adsorbed on its surface; the enzyme and polyvinylpyrrolidone were mixed at a mass ratio of 1:2, and then phosphate buffer was added, which was denoted as solution C; solution C was added to the PP@CaCO3 precipitate, and after stirring evenly, a calcium carbonate template PP@CaCO3@LDH solution with enzyme adsorbed on its surface was obtained, which was denoted as solution D; Step 3: Preparation of calcium carbonate template for surface-generated covalent organic framework microcapsules Ethanol solutions of trialdehyde resorcinol and benzidine were prepared at molar concentrations of 18.7 mmol / L and 28.2 mmol / L, respectively. The ethanol solutions of trialdehyde resorcinol and benzidine were simultaneously added to the PP@CaCO3@LDH solution obtained in step two to obtain solution E. In solution E, the molar concentration of trialdehyde resorcinol was 8.3 mmol / L and the molar concentration of benzidine was 12.5 mmol / L. Solution E was stirred for 5–45 min, the supernatant was removed, and the solution was centrifuged and washed with water to obtain a calcium carbonate template PP@CaCO3@LDH@TpBD with covalent organic framework microcapsules formed on its surface. Step 4: Preparation of enzymes immobilized in covalent organic framework microcapsules The calcium carbonate template obtained in step 3 for surface-generated covalent organic framework microcapsules was post-treated to remove the calcium carbonate template, and then centrifuged and washed with water; finally, the covalent organic framework microcapsule immobilized enzyme was obtained. In step two, the enzyme used is lactate dehydrogenase, which catalyzes the conversion of pyruvate into lactate.

2. The preparation method according to claim 1, characterized in that, In step four, the post-processing method involves dispersing the calcium carbonate template with covalent organic framework microcapsules formed on the surface in an ethylenediaminetetraacetic acid solution.

3. The covalent organic framework microencapsulated enzyme obtained by the preparation method according to any one of claims 1-2.

4. The covalent organic framework microencapsulated enzyme according to claim 3, characterized in that, The covalent organic framework microencapsulated enzyme has a particle size of 2.56-3.35 μm, with an atomic percentage of 76.35% carbon, 8.61% nitrogen, 14.49% oxygen, and 0.55% sulfur.

5. The application of the covalent organic framework microencapsulated enzyme according to claim 3, characterized in that, Enzymes immobilized in covalent organic framework microcapsules were used to catalyze the conversion of pyruvate to lactate, and the immobilized enzyme activity retained 70% of the free enzyme activity.

6. The application of the covalent organic framework microencapsulated enzyme according to claim 5, characterized in that, The covalent organic framework microcapsule immobilized enzyme retains 89% of its initial activity after being used 8 times.

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