Preparation method and application of conjugated polymer-enzyme-silicon oxide hybrid micro-capsule immobilized enzyme
Through the preparation method of conjugated polymer enzymes @enzymes@silica hybrid microcapsule type immobilization enzyme, the problem of low immobilization enzyme activity is solved, and enzyme immobilization with high activity, stability and recyclability is achieved, with relatively ideal industrial application prospects.
Patent Information
- Application Number
- CN202510175736.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-18
- Publication Date
- 2025-05-09
AI Technical Summary
The activity of existing immobilized enzymes is relatively low, making it difficult to maintain high enzyme activity and high stability.
The preparation method of conjugated polymer @enzyme @silicon oxide hybrid microcapsule type immobilized enzyme is adopted to form a microcapsule structure by combining the conjugated polymer and the silicon oxide material, providing an appropriate microenvironment for the enzyme and enhancing its stability and recyclability.
The activity, stability and recyclability of the enzyme were significantly improved, and the activity of the immobilized enzyme could reach up to 92.4% of the free enzyme, and more than 80% of the initial activity was retained after 8 recycles.
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Figure CN119955774A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to a method for preparing a microcapsule-type immobilized enzyme and an application thereof, and relates to a method for preparing a hybrid microcapsule-type immobilized enzyme for catalyzing pyruvic acid to generate lactic acid and an application thereof. Background Art
[0002] Enzymes are one of the most powerful natural catalysts in nature. They can show extremely high catalytic activity and selectivity under mild conditions. They are widely used in food, medicine, environmental protection, energy and other fields. They are the core of synthetic biology and green biomanufacturing. However, natural enzyme molecules have problems such as easy inactivation outside the cell, difficulty in separation, and low recycling rate. The development of enzyme immobilization technology has provided a broad space for enzymes to exert their efficient catalytic advantages and promoted the widespread application of enzymes in various fields of the chemical industry. However, the low activity of immobilized enzymes is still a major challenge facing current immobilization technology. Therefore, it is very important to develop an immobilization carrier that can maintain high enzyme activity and high stability. Summary of the invention
[0003] The present invention provides a method for preparing a conjugated polymer @ enzyme @ silicon oxide hybrid microcapsule type immobilized enzyme, which utilizes the advantages of excellent porosity, structural stability and abundant binding sites of conjugated polymers, combined with the good biocompatibility and chemical inertness of silicon oxide, to form a microcapsule structure that provides a suitable microenvironment for the enzyme on the basis of a high loading rate, thereby enhancing its stability and recyclability. Immobilizing the enzyme on the capsule wall can shorten the mass transfer distance, expose more reaction sites, and ultimately achieve higher immobilized enzyme activity, stability and recyclability. The present invention completes the construction of conjugated polymer @ enzyme @ silicon oxide on the surface of calcium carbonate microspheres through the Schiff base reaction of PEI and Tp and BD monomers, and biomimetic silicification on the surface of calcium carbonate, and obtains the conjugated polymer @ enzyme @ silicon oxide hybrid microcapsule type immobilized enzyme after removing the calcium carbonate by etching.
[0004] The purpose of the present invention is to provide a method for preparing a conjugated polymer @ enzyme @ silicon oxide hybrid microcapsule type immobilized enzyme. Compared with calcium carbonate @ conjugated polymer @ enzyme immobilized enzyme particles using surface adsorption, the preparation method of the conjugated polymer @ enzyme @ silicon oxide hybrid microcapsule type immobilized enzyme provided by the present invention combines the advantages of conjugated polymer and silicon oxide materials with the structural advantages of microcapsules, significantly enhancing the activity, stability and recyclability of the enzyme.
[0005] In order to achieve the above object, the present invention adopts the following technical solution: A method for preparing a conjugated polymer@enzyme@silica hybrid microcapsule-type immobilized enzyme comprises: using calcium carbonate doped with sodium polystyrene sulfonate PSS as a template, adding polyethyleneimine PEI to introduce a large number of amino groups on the surface of the calcium carbonate based on electrostatic interaction, adding 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde Tp and benzidine BD, and directionally generating a conjugated polymer on the surface of the calcium carbonate template through a Schiff base reaction; loading the enzyme on the surface of the conjugated polymer, adding polyethyleneimine again, inducing a Na2SiO3 solution to generate a silicon oxide layer on the surface of the conjugated polymer through its positive charge, and removing the calcium carbonate template through post-treatment to finally obtain a conjugated polymer@enzyme@silica hybrid microcapsule-type immobilized enzyme.
[0006] Preferably, the preparation method comprises the following steps: Step 1: Preparation of sodium polystyrene sulfonate-doped calcium carbonate template: Sodium polystyrene sulfonate was added to the CaCl2 solution, and after being mixed evenly, Na2CO3 solution was quickly added, and magnetic stirring was performed for 30 s and then precipitated for 15 min to obtain a calcium carbonate template doped with sodium polystyrene sulfonate. Step 2: Preparation of calcium carbonate@conjugated polymer particles: Adding polyethyleneimine to the sodium polystyrene sulfonate-doped calcium carbonate template obtained in step 1, stirring for a period of time and then centrifuging and washing with water to obtain a calcium carbonate template with polyethyleneimine adsorbed on the surface; adding an organic solution of 2,4,6-trihydroxybenzene-1,3,5-triformaldehyde Tp and benzidine BD to the calcium carbonate template with polyethyleneimine adsorbed on the surface, reacting for 60 min to obtain calcium carbonate@conjugated polymer; Step 3. Preparation of calcium carbonate@conjugated polymer@enzyme particles Adding a certain amount of enzyme solution to the aqueous solution of calcium carbonate@conjugated polymer particles obtained in step 2, stirring and adsorbing to obtain calcium carbonate@conjugated polymer@enzyme particles; Step 4: Preparation of calcium carbonate @ conjugated polymer @ enzyme @ silicon oxide particles Add polyethyleneimine to the calcium carbonate@conjugated polymer@enzyme particles obtained in step 3, stir for 20 min, then add to Na2SiO3 solution and stir for 30 min to induce the formation of a silicon oxide layer on the surface of the conjugated polymer to obtain calcium carbonate@conjugated polymer@enzyme@silicon oxide particles; Step 5. Preparation of conjugated polymer@enzyme@silica hybrid microcapsule-type immobilized enzyme The calcium carbonate@conjugated polymer@enzyme@silicon oxide particles obtained in step 4 are added to an EDTA solution to remove the calcium carbonate template, and the conjugated polymer@enzyme@silicon oxide hybrid microcapsule-type immobilized enzyme is obtained by centrifugation and washing.
[0007] Preferably, in step 1, the concentration of the CaCl2 solution is 0.33 mol / L.
[0008] Preferably, in step 1, the concentration of the Na2CO3 solution is 0.33 mol / L.
[0009] Preferably, in step 2, the molar ratio of 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde Tp to benzidine BD is 1:1.5.
[0010] Preferably, in step three, the enzyme used is lactate dehydrogenase, which can catalyze pyruvate into lactate.
[0011] Preferably, in step 4, the concentration of the Na2SiO3 solution is 20 mM.
[0012] The conjugated polymer@enzyme@silicon oxide hybrid microcapsule-type immobilized enzyme obtained by the preparation method is preferably immobilized in the capsule wall of the microcapsule, and the enzyme loading capacity reaches 102 mg / g, based on the mass of the carrier conjugated polymer TpBD.
[0013] Application: The conjugated polymer@enzyme@silicon oxide hybrid microcapsule-type immobilized enzyme prepared by the present invention is used to catalyze the conversion of pyruvate into lactic acid. The reaction is carried out for 30-40 min. The activity of the immobilized enzyme is calculated based on the reaction rate, and the maximum activity of the immobilized enzyme is 92.4% of the activity of the free enzyme. Moreover, after the microcapsule immobilized enzyme is recycled for 8 times, the activity of the immobilized enzyme can still reach more than 80% of the initial activity.
[0014] The advantages of the preparation method of the conjugated polymer @ enzyme @ silicon oxide hybrid microcapsule type immobilized enzyme proposed in the present invention are: the preparation process is green and mild, fast and simple, and the advantages of the excellent porosity, structural stability and rich binding sites of the conjugated polymer are utilized, combined with the good biocompatibility and chemical inertness of silicon oxide, and the formed microcapsule structure provides a suitable microenvironment for the enzyme on the basis of high loading rate; the enzyme is fixed on the capsule wall, which can shorten the mass transfer distance, expose more reaction sites, and finally achieve higher immobilized enzyme activity, stability and recyclability. By changing the monomer type, silicate concentration and template size in the preparation process, the physical structure and chemical microenvironment of the hybrid microcapsule can be regulated, and then microcapsule immobilized enzymes with different activities can be obtained. Compared with calcium carbonate @ conjugated polymer @ enzyme particle immobilized enzyme, the reaction rate of the substrate catalyzed by the conjugated polymer @ enzyme @ silicon oxide hybrid microcapsule type immobilized enzyme prepared by the present invention is significantly improved, and the immobilized enzyme activity retains up to 92.4% of the free enzyme, and is easy to separate and recycle, and retains more than 80% of the initial immobilized enzyme activity after 8 cycles. The activity, stability and recyclability of the immobilized enzyme are significantly improved, and it has an ideal prospect for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is a scanning electron microscope image of the calcium carbonate@conjugated polymer@enzyme particles prepared in Comparative Example 1; Figure 2 This is a scanning electron micrograph of the conjugated polymer@enzyme@silicon oxide hybrid microcapsule-type immobilized enzyme prepared in Example 1; Figure 3 This is a comparison chart of the reaction rates during use of the immobilized enzymes prepared in Comparative Example 1 and Example 1; Figure 4 This is a comparison chart of the reaction rates during use of the conjugated polymer@enzyme@silicon oxide hybrid microcapsule-type immobilized enzymes prepared in Example 1, Example 2, Example 3, Example 4 and Example 5; Figure 5 This is a graph showing the relative activity of the conjugated polymer@enzyme@silica hybrid microcapsule-type immobilized enzyme prepared in Example 4 during cyclic use. DETAILED DESCRIPTION
[0016] The present invention proposes a method for preparing a conjugated polymer @ enzyme @ silicon oxide hybrid microcapsule type immobilized enzyme. The design idea is: utilizing the advantages of excellent porosity, structural stability and abundant binding sites of conjugated polymers, combined with the good biocompatibility and chemical inertness of silicon oxide, the formed microcapsule structure provides a suitable microenvironment for the enzyme on the basis of high loading rate; the enzyme is fixed on the capsule wall, which can shorten the mass transfer distance, expose more reaction sites, and finally achieve higher immobilized enzyme activity, stability and recyclability. Its basic scheme is: using electrostatic interaction to adsorb polyethyleneimine on the surface of calcium carbonate template to introduce a large number of amino groups, adding 2,4,6-trihydroxybenzene-1,3,5-triformaldehyde and benzidine to generate conjugated polymer on the surface of calcium carbonate template by Schiff base reaction; adsorbing the enzyme on the surface of conjugated porous polymer, and then using the positive charge of polyethyleneimine to induce silicate to generate a silicon oxide layer on the surface of conjugated porous polymer, and removing the calcium carbonate template after post-treatment, finally obtaining a conjugated polymer @ enzyme @ silicon oxide hybrid microcapsule type immobilized enzyme.
[0017] The enzyme loading amount in the following examples and comparative examples is specifically the enzyme loading amount calculated based on the mass of the carrier conjugated polymer TpBD.
[0018] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings, comparative examples and specific embodiments. The described specific embodiments are only used to explain the present invention and are not intended to limit the present invention.
[0019] Comparative Example 1 The steps of preparing a calcium carbonate@conjugated polymer@enzyme particle immobilized enzyme are as follows: Step 1, preparation of calcium carbonate template doped with sodium polystyrene sulfonate; prepare 10 mL of 0.33 mol / L CaCl2 solution, add 30 mg of sodium polystyrene sulfonate to it to make the final concentration of 3.0 mg / mL, stir evenly, and then quickly add 10 mL of 0.33 mol / L Na2CO3 solution, stir magnetically for 30 s and then precipitate for 15 min, the resulting precipitate is calcium carbonate template doped with sodium polystyrene sulfonate; Step 2, preparation of calcium carbonate @ conjugated polymer particles; add 1 / 5 of the mass of the calcium carbonate template in step 1 to 10 mL of a polyethyleneimine solution with a concentration of 1 mg / mL and a pH of 7, stir for 30 minutes, and then centrifuge and wash with water to obtain a calcium carbonate template with polyethyleneimine adsorbed on the surface; dissolve 2,4,6-trihydroxybenzene-1,3,5-triformaldehyde Tp and benzidine BD in 4 mL of N,N-dimethylformamide solution at a molar ratio of 1:1.5, respectively, to make the final concentrations of 8 mmol / L and 12 mmol / L, respectively, and then add them to the calcium carbonate template with polyethyleneimine adsorbed on the surface, and react for 60 min to obtain calcium carbonate @ conjugated polymer particles; Step 3, preparation of calcium carbonate @ conjugated polymer @ enzyme particles; add 140 μL of 1 mg / mL enzyme solution to 7 mL of 27 mg / mL calcium carbonate @ conjugated polymer particle aqueous solution obtained in step 2, and stir to obtain calcium carbonate @ conjugated polymer @ enzyme particles after adsorption; 189.14 mg of calcium carbonate@conjugated polymer@enzyme particles obtained in Comparative Example 1 were dispersed in 7 mL of 50 mM phosphate buffer at pH 7, 1 mL was taken and then 0.5 mL of 10 mM pyruvic acid solution and reduced nicotinamide adenine dinucleotide NADH solution were added at 35°C for catalytic reaction. The reaction ratio of NADH to pyruvic acid in the catalytic process was 1:1. Therefore, the change in the content of the reactant reduced nicotinamide adenine dinucleotide NADH was detected by a UV-visible spectrophotometer, and the immobilized enzyme activity was calculated by the reaction rate in the first 5 min.
[0020] The enzyme loading was measured to be 102 mg / g, and the reaction rate of catalyzing the conversion of pyruvate to lactate was 0.186 mM min -1 .
[0021] The activity of the immobilized enzyme was calculated based on the concentration of the enzyme-catalyzed substrate per unit time. After calculation, the activity of the immobilized enzyme prepared in Comparative Example 1 was 38.6% of that of the free enzyme.
[0022] Example 1 The steps for preparing the conjugated polymer@enzyme@silica hybrid microcapsule-type immobilized enzyme are as follows: Step 1: Preparation of sodium polystyrene sulfonate-doped calcium carbonate template: Prepare 10 mL of 0.33 mol / L CaCl2 solution, add 30 mg of sodium polystyrene sulfonate to make the final concentration 3.0 mg / mL, mix well, quickly add 10 mL of 0.33 mol / L Na2CO3 solution, stir magnetically for 30 s and then precipitate for 15 min to obtain a calcium carbonate template doped with sodium polystyrene sulfonate; Step 2: Preparation of calcium carbonate@conjugated polymer particles: Take 1 / 5 of the mass of the calcium carbonate template in step 1 and add it to 10 mL of polyethyleneimine solution with a concentration of 1 mg / mL and a pH of 7, stir for 30 minutes, then centrifuge and wash with water to obtain a calcium carbonate template with polyethyleneimine adsorbed on the surface; dissolve 2,4,6-trihydroxybenzene-1,3,5-triformaldehyde Tp and benzidine BD in 4 mL of N,N-dimethylformamide solution at a molar ratio of 1:1.5, respectively, to make the final concentrations of 8 mmol / L and 12 mmol / L, respectively, and then add them to the calcium carbonate template with polyethyleneimine adsorbed on the surface, and react for 60 min to obtain calcium carbonate@conjugated polymer; Step 3: Preparation of calcium carbonate@conjugated polymer@enzyme particles: Add 140 μL of 1 mg / mL enzyme solution to 7 mL of 27 mg / mL calcium carbonate@conjugated polymer particle aqueous solution obtained in step 2, and stir to obtain calcium carbonate@conjugated polymer@enzyme particles after adsorption; Step 4: Preparation of calcium carbonate @ conjugated polymer @ enzyme @ silicon oxide particles Add 10 mL of 2 mg / mL, pH 7 polyethyleneimine solution to the calcium carbonate@conjugated polymer@enzyme particles obtained in step 3, stir for 20 min, then add 10 mL of 50 mM Na2SiO3 solution, continue stirring for 30 min, and induce the formation of a silicon oxide layer on the surface to obtain calcium carbonate@conjugated polymer@enzyme@silicon oxide particles; Step 5. Preparation of conjugated polymer@enzyme@silica hybrid microcapsule-type immobilized enzyme The calcium carbonate@conjugated polymer@enzyme@silica particles obtained in step 4 were added to 7 mL of 20 mM EDTA solution with a pH of 6.5 to remove the calcium carbonate template, and the conjugated polymer@enzyme@silica hybrid microcapsule-type immobilized enzyme was obtained by centrifugation and washing.
[0023] 195 mg of the conjugated polymer@enzyme@silica hybrid microcapsule immobilized enzyme obtained in Example 1 was dispersed in 7 mL of 50 mM phosphate buffer at pH 7, 1 mL was added with 0.5 mL of 10 mM pyruvic acid solution and reduced nicotinamide adenine dinucleotide NADH solution at 35°C for catalytic reaction, the change in the content of the reactant reduced nicotinamide adenine dinucleotide NADH was detected by a UV-visible spectrophotometer, and the activity of the immobilized enzyme was calculated by recording the reaction rate in the first 5 min.
[0024] The enzyme loading was measured to be 102 mg / g, and the reaction rate of catalyzing the conversion of pyruvate to lactate was 0.345 mM min -1 , the activity of the immobilized enzyme was 66.3% of that of the free enzyme.
[0025] Example 2 Preparation of conjugated polymer@enzyme@silica (sodium silicate 40mM) hybrid microcapsule immobilized enzyme, Example 2 is basically the same as Example 1, except that: during the operation of step 4, the concentration of the added Na2SiO3 solution is 40mM. The enzyme loading was measured to be 102 mg / g, and the initial reaction rate was 0.349 mM min -1 , the activity of the immobilized enzyme was 67.1% of that of the free enzyme.
[0026] Example 3 Preparation of conjugated polymer@enzyme@silica (sodium silicate 30mM) hybrid microcapsule immobilized enzyme, Example 3 is basically the same as Example 1, except that: during the operation of step 4, the concentration of the added Na2SiO3 solution is 30mM. The enzyme loading was measured to be 102 mg / g, and the initial reaction rate was 0.434 mM min -1 , the activity of the immobilized enzyme was 83.4% of that of the free enzyme.
[0027] Example 4 Preparation of conjugated polymer@enzyme@silica (sodium silicate 20mM) hybrid microcapsule immobilized enzyme, Example 4 is basically the same as Example 1, except that: during the operation of step 4, the concentration of the added Na2SiO3 solution is 20mM. The enzyme loading was measured to be 102 mg / g, and the initial reaction rate was 0.480 mM min -1 , the activity of the immobilized enzyme was 92.3% of that of the free enzyme.
[0028] Example 5 Preparation of conjugated polymer@enzyme@silica (10 mM sodium silicate) hybrid microcapsule-type immobilized enzyme, Example 5 is basically the same as Example 1, except that: during the operation of step 4, the concentration of the added Na2SiO3 solution is 10 mM. The enzyme loading was measured to be 102 mg / g, and the initial reaction rate was 0.385 mM min -1 , the activity of the immobilized enzyme was 74.0% of that of the free enzyme.
[0029] The concentration of the Na2SiO3 solution that generates the silicon oxide protective layer will affect the density and thickness of the silicon oxide protective layer. The above structural differences will affect the enrichment and transfer process of the substrate, resulting in different catalytic reaction rates. In summary, through the comparison chart of the reaction rates of immobilized enzymes corresponding to the above Examples 1-5, it is found that the conjugated polymer @ enzyme @ silicon oxide (sodium silicate 20mM) hybrid microcapsule-type immobilized enzyme obtained when the concentration of sodium silicate induced to generate silicon oxide is 20 mM finally makes the enrichment-transfer process of the substrate reach the best balance and the performance is optimal. The conjugated polymer @ enzyme @ silicon oxide hybrid microcapsule-type immobilized enzyme finally prepared by the present invention retains up to 92.4% of the activity of the free enzyme, and the activity of the immobilized enzyme after 8 cycles is more than 80% of the initial immobilized enzyme activity. It is used to catalyze the conversion of pyruvate to lactic acid, which can significantly improve the utilization value and reduce production costs.
[0030] In the conjugated polymer@enzyme@silica hybrid microcapsules prepared by the present invention: (1) The conjugated polymer plays the role of immobilizing the enzyme, and after the enzyme is immobilized, the silicon oxide layer is covered to protect it. Because the enzyme immobilization amount is calculated based on the mass of the carrier conjugated polymer TpBD, the synthetic steps and amounts in the comparative example and the early stage of the example are the same, so the calculated immobilization amount is the same, which has nothing to do with the different masses of the silicon oxide layer.
[0031] (2) The biggest difference between the comparative example and the example is that the comparative example is a particle while the example is a microcapsule, and the biggest difference between the examples is the concentration of the Na2SiO3 solution used to generate the silicon oxide protective layer. This will affect the density and thickness of the silicon oxide protective layer, and is also a structural difference. The above structural differences will affect the enrichment and transfer of the substrate, resulting in different catalytic reaction rates. If the concentration is too low, the enrichment effect is poor, and if the concentration is too high, the mass transfer effect is poor. The silicon oxide protective layer induced by the Na2SiO3 solution with a concentration of 20 mM enables the enrichment-transfer process of the substrate to reach the best balance, so the catalytic reaction rate is the best.
[0032] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. A method for preparing a conjugated polymer@enzyme@silicon oxide hybrid microcapsule type immobilized enzyme, characterized in that: Using calcium carbonate doped with sodium polystyrene sulfonate (PSS) as a template, polyethyleneimine (PEI) was added to introduce a large number of amino groups on the surface of calcium carbonate based on electrostatic interaction. 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde (Tp) and benzidine (BD) were added to form a conjugated polymer on the surface of the calcium carbonate template through a Schiff base reaction. The enzyme was loaded on the surface of the conjugated polymer, and polyethyleneimine was added again to induce Na2SiO3 solution to form a silicon oxide layer on the surface of the conjugated polymer through its positive charge. The calcium carbonate template was removed through post-treatment to finally obtain a conjugated polymer@enzyme@silica hybrid microcapsule-type immobilized enzyme.
2. The preparation method according to claim 1, characterized in that: The following steps are involved: Step 1: Preparation of sodium polystyrene sulfonate-doped calcium carbonate template: Sodium polystyrene sulfonate was added to the CaCl2 solution, and after being mixed evenly, Na2CO3 solution was quickly added, and magnetic stirring was performed for 30 s and then precipitated for 15 min to obtain a calcium carbonate template doped with sodium polystyrene sulfonate. Step 2: Preparation of calcium carbonate@conjugated polymer particles: Adding polyethyleneimine to the sodium polystyrene sulfonate-doped calcium carbonate template obtained in step 1, stirring for a period of time and then centrifuging and washing with water to obtain a calcium carbonate template with polyethyleneimine adsorbed on the surface; adding an organic solution of 2,4,6-trihydroxybenzene-1,3,5-triformaldehyde Tp and benzidine BD to the calcium carbonate template with polyethyleneimine adsorbed on the surface, reacting for 60 min to obtain calcium carbonate@conjugated polymer; Step 3. Preparation of calcium carbonate@conjugated polymer@enzyme particles Adding a certain amount of enzyme solution to the aqueous solution of calcium carbonate@conjugated polymer particles obtained in step 2, stirring and adsorbing to obtain calcium carbonate@conjugated polymer@enzyme particles; Step 4: Preparation of calcium carbonate @ conjugated polymer @ enzyme @ silicon oxide particles Add polyethyleneimine to the calcium carbonate@conjugated polymer@enzyme particles obtained in step 3, stir for 20 min, then add to Na2SiO3 solution and stir for 30 min to induce the formation of a silicon oxide layer on the surface of the conjugated polymer to obtain calcium carbonate@conjugated polymer@enzyme@silicon oxide particles; Step 5. Preparation of conjugated polymer@enzyme@silica hybrid microcapsule-type immobilized enzyme The calcium carbonate@conjugated polymer@enzyme@silicon oxide particles obtained in step 4 are added to an EDTA solution to remove the calcium carbonate template, and the conjugated polymer@enzyme@silicon oxide hybrid microcapsule-type immobilized enzyme is obtained by centrifugation and washing.
3. The preparation method according to claim 2, characterized in that: In step 1, the concentration of the CaCl2 solution is 0.33 mol / L.
4. The preparation method according to claim 2, characterized in that: In step 1, the concentration of the Na2CO3 solution is 0.33 mol / L.
5. The preparation method according to claim 2, characterized in that: In step 2, the molar ratio of 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde Tp to benzidine BD is 1:1.
5.
6. The preparation method according to claim 2, characterized in that: In step three, the enzyme used is lactate dehydrogenase, which can catalyze pyruvate into lactate.
7. The preparation method according to claim 2, characterized in that: In step 4, the concentration of the Na2SiO3 solution is 20 mM.
8. The conjugated polymer@enzyme@silicon oxide hybrid microcapsule type immobilized enzyme obtained according to the preparation method according to any one of claims 1 to 7.
9. The conjugated polymer@enzyme@silicon oxide hybrid microcapsule type immobilized enzyme according to claim 8, characterized in that: The enzyme molecules are fixed in the capsule wall of the microcapsule, and the enzyme loading capacity reaches 102 mg / g, based on the mass of the carrier conjugated polymer TpBD.
10. Use of the conjugated polymer@enzyme@silicon oxide hybrid microcapsule-type immobilized enzyme according to claim 8 in catalyzing the production of lactic acid from pyruvate.