Immobilized enzyme catalyst based on bifunctional covalent organic framework as well as preparation method and application of immobilized enzyme catalyst

Abstract

The invention discloses an immobilized enzyme catalyst based on a bifunctional covalent organic framework. An EBCOF nanosheet functionalized based on a quaternary ammonium group has dual functions of enzyme loading and silicification induction. The preparation method mainly comprises the following steps: firstly, adsorbing enzyme on the surface of the EBCOF nanosheet through electrostatic interaction, and then inducing a silicon oxide coating to be formed on the surface of the EBCOF nanosheet in situ through a quaternary ammonium group on the EBCOF nanosheet. The ultrathin EBCOF nanosheet has high enzyme loading capacity, and 3.4 milligrams of enzyme can be adsorbed by each milligram of the EBCOF nanosheet. And meanwhile, the formed silicon oxide coating effectively protects enzyme molecules. Compared with direct adsorption of the enzyme on the EBCOF, the existence of the silicon oxide coating improves the temperature, pH and cycling stability of the immobilized enzyme. The method can be expanded to construction of an immobilized multi-enzyme catalytic system. Compared with a free enzyme system, the initial activity of the immobilized multienzyme catalytic system is improved by 143%.

Description

Technical Field

[0001] The invention belongs to the technical field of immobilized enzymes, and in particular relates to an immobilized enzyme method based on a bifunctional covalent organic framework and an application thereof. Background Art

[0002] Enzymes are highly active and selective biocatalysts that can catalyze reactions in a green, efficient and mild manner. They are widely used in the fields of medicine, chemical industry, carbon dioxide conversion, etc. However, the poor stability of free enzymes in harsh environments (such as high temperature, strong acid / base), high cost, and difficulty in recovery limit their industrial application. Immobilized enzyme technology fixes enzymes on solid carriers by adsorption, embedding, covalent bonding, and cross-linking, which improves the stability of enzymes under extreme conditions and enables the reuse of enzymes. Covalent organic frameworks (COFs) are an emerging class of crystalline porous organic polymers with the advantages of high specific surface area, adjustable functions, and stable structure, providing excellent conditions for immobilized enzymes. It is very promising to rationally design immobilized enzyme catalysts based on COFs to have high enzyme loading and excellent stability. Summary of the invention

[0003] In view of the above-mentioned prior art, the present invention provides an enzyme immobilization method based on a bifunctional covalent organic framework and its application. In the present invention, the covalent organic framework nanosheet prepared from ethidium bromide and trialdehyde phloroglucinol is referred to as EBCOF nanosheet, which has the dual functions of adsorbing enzymes and inducing silicification. This simple and effective enzyme immobilization strategy has a higher enzyme loading capacity and improves the stability of the enzyme.

[0004] In order to solve the above technical problems, the present invention proposes an immobilized enzyme catalyst based on a bifunctional covalent organic framework, wherein a covalent organic framework (EBCOF) nanosheet is prepared from ethidium bromide and trialdehyde phloroglucinol; the obtained covalent organic framework nanosheet has the dual functions of loading enzyme and inducing silicification, and the method of immobilizing enzyme firstly adsorbs the enzyme on the surface of the ethidium bromide covalent organic framework nanosheet through electrostatic interaction, and then the quaternary ammonium group on the covalent organic framework nanosheet induces the in-situ formation of a silicon oxide coating on its surface. The preparation method of the immobilized enzyme catalyst comprises the following specific steps:

[0005] Step 1) preparing a covalent organic framework nanosheet dispersion: dispersing ethidium bromide and trialdehyde phloroglucinol in equal volumes of dimethyl sulfoxide solution at a molar ratio of ethidium bromide to trialdehyde phloroglucinol of 3:2, and uniformly dispersing by ultrasonication to obtain ethidium bromide dispersion and trialdehyde phloroglucinol dispersion, respectively; dropping the trialdehyde phloroglucinol dispersion into the ethidium bromide dispersion, and slowly stirring to obtain a mixed solution; allowing the mixed solution to react at room temperature, diluting it with deionized water, and dialyzing it in water with a dialysis bag with a molecular weight cutoff of 30,000 Da to obtain a covalent organic framework nanosheet dispersion;

[0006] Step 2) preparing enzyme / covalent organic framework: mixing the covalent organic framework nanosheet dispersion obtained in step 1) with the enzyme solution, wherein the mass ratio of the enzyme to the covalent organic framework nanosheet is 0.5-20:1; shaking on a shaker at 100-200 rpm for 10-60 min to obtain an enzyme / covalent organic framework dispersion;

[0007] Step 3) Preparation of immobilized enzyme catalyst based on covalent organic framework: prepare a molar concentration of 10-60 mmol L -1 sodium silicate solution, adjusting the solution pH to 7-8 with hydrochloric acid; adding the sodium silicate solution to the enzyme / covalent organic framework dispersion obtained in step 2) at a volume ratio of 1:1, mixing on a shaker, removing the supernatant by centrifugation, washing with PBS buffer, and obtaining a precipitate of enzyme / covalent organic framework immobilized enzyme with a silicon oxide coating grown on the surface.

[0008] Furthermore, the preparation method of the present invention, wherein:

[0009] The enzyme is one or more of alcohol dehydrogenase (ADH), alcohol oxidase (AOX), catalase (CAT) and glycolaldehyde synthase (GALS).

[0010] In step 1), the reaction time at room temperature is 24 hours; and the dialysis time is 3 days.

[0011] In step 3), the shaking speed is 100-200 rpm for 10-60 min; the speed of centrifugal removal of supernatant is 7000-10000 rpm for 2-5 min; the concentration of PBS buffer is 50 mmol L -1 , pH was 7.5, and the number of washings was 3 times.

[0012] The immobilized enzyme catalyst based on the bifunctional covalent organic framework prepared by the present invention is used to realize the conversion of methanol to ethanolaldehyde. 100mmol L -1 Methanol can obtain 3.8mmol L -1ethanolaldehyde, and the initial activity of the immobilized enzyme catalyst was increased by 143% compared with the free enzyme.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] Compared with the prior art, the enzyme immobilization method based on the bifunctional covalent organic framework material proposed in the present invention first adsorbs the enzyme on the surface of the EBCOF nanosheet through electrostatic interaction, and then the EBCOF nanosheet induces sodium silicate to silicify on its surface to form a silicon oxide coating, which further protects the fragile enzyme molecules. The ultrathin EBCOF nanosheet has a highly accessible surface and more exposed adsorption sites, and each milligram of EBCOF nanosheet can adsorb 3.4 milligrams of enzyme. At the same time, the quaternary ammonium group induces the formation of the silicon oxide coating on the surface of the EBCOF nanosheet, and the presence of the silicon oxide coating improves the temperature, pH and cyclic stability of the immobilized enzyme. This adsorption-silicification immobilized enzyme strategy based on a bifunctional covalent organic framework effectively improves the enzyme loading and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a scanning electron microscope image of the EBCOF nanosheet in Example 1;

[0016] Figure 2 is a scanning electron microscope image of ADH / EBCOF in Example 1;

[0017] Figure 3 This is a scanning electron microscope image of ADH / EBCOF@Silica prepared in Example 1;

[0018] Figure 4 To optimize the loading amount of different enzymes in the preparation of ADH / EBCOF by the method of the present invention;

[0019] Figure 5 Comparison of the catalytic activities of free ADH in comparative example 1, ADH / EBCOF in example 2, and ADH / EBCOF@Silica in example 1;

[0020] Figure 6 The pH stability comparison of free ADH in comparative example 1, ADH / EBCOF in example 2, and ADH / EBCOF@Silica in example 1;

[0021] Figure 7 The temperature stability comparison of free ADH in comparative example 1, ADH / EBCOF in example 2, and ADH / EBCOF@Silica in example 1;

[0022] Figure 8 Comparison of the cycle stability of ADH / EBCOF in Example 2 and ADH / EBCOF@Silica in Example 1;

[0023] Fig. 9 Comparison of the catalytic activities of free AOX&CAT&GALS in comparative example 2 and (AOX&CAT&GALS) / EBCOF@Silica in example 5. DETAILED DESCRIPTION

[0024] The present invention proposes an enzyme immobilization method based on a bifunctional covalent organic framework. The design idea is that the EBCOF nanosheets prepared by ethidium bromide and trialdehyde phloroglucinol have the dual functions of loading enzymes and inducing silicification. The method first adsorbs the enzyme on the surface of the EBCOF nanosheet through electrostatic interaction, and then the EBCOF nanosheet induces sodium silicate to mineralize on its surface to form a silicon oxide coating. The ultrathin EBCOF nanosheet has a high enzyme loading capacity, and each milligram of EBCOF nanosheet can adsorb 3.4 milligrams of enzyme. At the same time, the formed silicon oxide coating effectively protects the enzyme molecules. Compared with directly adsorbing the enzyme on EBCOF, the presence of the silicon oxide coating improves the temperature, pH and cyclic stability of the immobilized enzyme. In addition, in view of the high enzyme loading capacity of the EBCOF nanosheet, it can be extended to multi-enzyme catalysis and an immobilized multi-enzyme catalytic system is constructed. Compared with the free enzyme system, the initial activity of the immobilized multi-enzyme catalytic system is increased by 143%. The method provides a promising method for immobilizing enzymes and expands the application of COF in the field of enzyme catalysis.

[0025] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the following embodiments are by no means intended to limit the present invention in any way.

[0026] Example 1: Preparation of ADH / EBCOF immobilized enzyme

[0027] Step 1) Preparation of EBCOF nanosheet dispersion

[0028] 0.3mmol EB and 0.2mmol TFP were dispersed in 5mL dimethyl sulfoxide, ultrasonically dispersed for 0.5h, and EB dispersion and TFP dispersion were obtained respectively; TFP dispersion was added dropwise to EB dispersion, and slowly stirred to mix evenly, and then the mixed solution was allowed to stand at room temperature for 24h. The reacted solution was diluted with an equal volume of deionized water, and then dialyzed in water for 3d with a dialysis bag with a molecular weight cutoff of 30000Da to remove small molecules and oligomers, and finally EBCOF nanosheet dispersion was obtained. Figure 1 The scanning electron microscope image of the EBCOF nanosheets in the dispersion shows that the EBCOF nanosheets are ultra-thin sheet structures with smooth surface morphology.

[0029] Step 2) Preparation of ADH / EBCOF:

[0030] Take alcohol dehydrogenase (ADH) as the model enzyme and take 45 μL of the solution obtained in step 1) with a concentration of 4.4 mg mL -1 100 μL of EBCOF nanosheet dispersion with a concentration of 1 mg mL -1 ADH solution was mixed (the mass ratio of enzyme to EBCOF was 1:2) and PBS buffer (50 mmol L -1 , pH 7.5) to 1 mL. Shake at 120 rpm for 30 min on a shaker, centrifuge (9000 rpm, 3 min) to remove the supernatant, and wash with PBS (50 mmol L -1 , pH 7.5) buffer solution for three times to obtain an ADH / EBCOF dispersion, in which the precipitate was ADH / EBCOF, which was freeze-dried and stored at 4°C. Figure 2 is a scanning electron microscope image of the ADH / EBCOF, and Figure 1 Compared with the ADH / EBCOF, no morphological changes occurred.

[0031] Step 3) Preparation of ADH / EBCOF@Silica

[0032] An equal volume of sodium silicate (60 mmol L -1 , pH 7.5) solution, shaken on a shaker at 120 rpm for 10 min, centrifuged (9000 rpm, 3 min) to remove the supernatant, and washed with PBS buffer (50 mmol L -1 The obtained precipitate was an enzyme / covalent organic framework-immobilized enzyme catalyst with a silicon oxide coating grown on the surface, denoted as ADH / EBCOF@Silica, and was freeze-dried and stored at 4°C. Figure 3 This is a scanning electron microscope image of the ADH / EBCOF@Silica prepared in this example. Figure 1 and Figure 2 In comparison, ADH / EBCOF@Silica has a rougher surface morphology, indicating that EBCOF induces silicification and forms a silicon oxide coating.

[0033] Example 2: Catalytic activity, pH, temperature and cycle stability test of ADH / EBCOF:

[0034] ADH / EBCOF containing 0.1 mg ADH prepared according to steps 1-2 of Example 1 was dispersed in 800 μL PBS buffer (50 mmol L -1 , pH 7.5), and then 100 μL of ethanolaldehyde solution (100 mmol L -1 ) and 100 μL reduced coenzyme I (NADH) solution (100 mmol L -1) triggers the catalytic reaction, catalyzing the conversion of ethanolaldehyde into ethylene glycol. Then, the reaction solution was shaken in a 35°C water bath, and 100 μL of the reaction solution was taken out at each time point and 100 μL of sulfuric acid solution (1 mmol L -1 ) to terminate the reaction. The concentration of ethylene glycol was detected by high performance liquid chromatography (HPLC). Figure 5 shown.

[0035] According to the ADH / EBCOF sample containing 0.1 mg ADH and 0.2 mg EBCOF prepared in step 1-2 of Example 1, 6 samples were incubated at room temperature with pH values ​​of 3, 5, 7.5, 9, 11, and 12 for 30 min, and 3 samples were incubated at 30° C., 40° C., and 50° C. with pH value of 7.5 for 30 min. Then, all the above samples were reacted under the same reaction conditions (pH value of 7.5 and temperature of 35° C.) to test the initial catalytic activity, and the relative activity under different conditions was obtained by comparative calculation ( Figure 6 and Figure 7 ).

[0036] The cycle stability test conditions are consistent with the catalytic activity test conditions. After 2 minutes of reaction, the supernatant is centrifuged to test the ethylene glycol content and calculate the initial catalytic activity. Then, the ADH / EBCOF precipitate is washed and centrifuged to recover for the next cycle reaction. Finally, the relative activity is obtained by comparison, such as Figure 8 shown.

[0037] Comparative Example 1: Catalytic activity and pH and temperature stability test of free ADH

[0038] 100 μL of ADH solution (1 mg mL -1 ), 100 μL glycolaldehyde solution (100 mmol L -1 ) and 100 μL NADH solution (100 mmol L -1 ) and diluted to 1 mL for reaction. Then, the reaction solution was shaken in a 35°C water bath, and 100 μL of the reaction solution was taken out at each time point and 100 μL of sulfuric acid solution (1 mmol L -1 ) to terminate the reaction. The concentration of ethylene glycol was detected by high performance liquid chromatography (HPLC). Figure 5 shown.

[0039] ADH was incubated at room temperature at different pH values ​​(3, 5, 7.5, 9, 11, 12) and at pH 7.5 at different temperatures (30°C, 40°C, 50°C) for 30 min, and then the initial catalytic activity was tested under the same reaction conditions (pH 7.5, temperature 35°C). The relative activity under different conditions was calculated and compared. Figure 6 and Figure 7 ).

[0040] Example 3: Optimization of enzyme loading of EBCOF

[0041] Eight ADH / EBCOF samples containing 0.2 mg EBCOF and different enzyme amounts (0.1 mg, 0.5 mg, 1 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg and 4.0 mg, respectively) were prepared according to steps 1-2 of Example 1. The enzyme concentration in the supernatant was determined by the Coomassie Brilliant Blue method to calculate the enzyme loading per mg EBCOF. Figure 4 Figure 2 shows the enzyme loading of EBCOF at different enzyme dosages. When the enzyme dosage is 2 mg, the enzyme loading of EBCOF tends to be balanced. When the enzyme dosage is 4 mg, the loading of EBCOF nanosheets reaches 3.4 mg. -1 .

[0042] Example 4: Preparation of ADH / EBCOF@Silica and Testing of Catalytic Activity and pH, Temperature and Cyclic Stability

[0043] The ADH / EBCOF@Silica prepared according to the preparation method of Example 1 was dispersed in 800 μL PBS buffer (50 mmol L -1 , pH 7.5), and then 100 μL of ethanolaldehyde solution (100 mmol L -1 ) and 100 μL NADH solution (100 mmol L -1 ) triggers the catalytic reaction, catalyzing the conversion of ethanolaldehyde into ethylene glycol. Then, the reaction solution was shaken in a 35°C water bath, and 100 μL of the reaction solution was taken out at each time point and 100 μL of sulfuric acid solution (1 mmol L -1 ) to terminate the reaction. The concentration of ethylene glycol was detected by high performance liquid chromatography (HPLC). Figure 5 As shown, by comparing the catalytic activities of ADH / EBCOF@Silica and ADH / EBCOF, it is shown that the effect of silica coating on mass transfer is negligible.

[0044] Six samples of ADH / EBCOF@Silica prepared according to the preparation method of Example 1 were incubated at room temperature with pH values ​​of 3, 5, 7.5, 9, 11, and 12 for 30 min, and three samples were incubated at 30°C, 40°C, and 50°C with pH value of 7.5 for 30 min. All the samples were then tested for initial catalytic activity under the same reaction conditions (pH value of 7.5 and temperature of 35°C). The relative activities under different conditions were calculated and compared ( Figure 6 and Figure 7 ).

[0045] The cycle stability test conditions are consistent with the catalytic activity test conditions. After 2 minutes of reaction, the supernatant is centrifuged to test the ethylene glycol content and calculate the initial catalytic activity. Then, the ADH / EBCOF@Silica precipitate is washed and centrifuged to recover for the next cycle reaction. The relative activity is finally compared, as shown in Figure 8 shown.

[0046] like Figure 6 , Figure 7 and Figure 8 As shown, at pH 11, 50°C and after 5 cycles of treatment, the stability of ADH / EBCOF@Silica was improved by 165%, 113% and 44% compared with ADH / EBCOF, respectively, indicating that the presence of the silica coating effectively improved the stability of the enzyme.

[0047] Example 5: Preparation and catalytic activity test of (AOX&CAT&GALS) / EBCOF@Silica

[0048] In view of the high enzyme loading of EBCOF nanosheets, the method of the present invention was extended to multi-enzyme catalysis, and an immobilized multi-enzyme catalytic system was constructed by this method. Immobilized alcohol oxidase (AOX), catalase (CAT) and glycolaldehyde synthase (GALS) catalyzed the conversion of methanol to glycolaldehyde. Specifically, AOX catalyzed the conversion of methanol to formaldehyde and produced H2O2 at the same time, and CAT was used to remove toxic H2O2; then GALS catalyzed the conversion of formaldehyde to glycolaldehyde.

[0049] 1) Preparation of AOX&CAT&GALS) / EBCOF@Silica

[0050] 100 μL AOX solution (8 mg mL -1 ), 100 μL CAT solution (6 mg mL -1 ), 625 μL GALS solution (4.8 mg mL -1 ) and 540 μL EBCOF solution (4.4 mg mL -1 ) and washed with PBS buffer (50 mmol L -1 , pH 7.5) to 6 mL, and then shaken at 120 rpm for 30 min. Next, 6 mL of sodium silicate solution (60 mmol L-1, pH 7.5) was added to the above solution for 10 min, centrifuged (9000 rpm, 3 min) to remove the supernatant, and washed with PBS buffer (50 mmol L-1). -1 , pH 7.5) for three times, and the resulting precipitate was (AOX&CAT&GALS) / EBCOF@Silica.

[0051] 2) Catalytic activity test of (AOX&CAT&GALS) / EBCOF@Silica

[0052] The (AOX&CAT&GALS) / EBCOF@Silica prepared above was dispersed in 850 μL PBS buffer (50 mmol / L -1 , pH 7.5), and then 50 μL of methanol solution (2 mol L -1 )、50μL magnesium sulfate solution (200mmol L -1 ) and 50 μL thiamine pyrophosphate (TPP) solution were used to trigger the catalytic reaction, catalyzing the conversion of methanol into ethanolaldehyde. Then, the reaction solution was shaken in a 35°C water bath, and 100 μL of the reaction solution was taken out at each time point and 100 μL of sulfuric acid solution (1 mmol L -1 ) to terminate the reaction. The concentration of glycolaldehyde was detected by high performance liquid chromatography (HPLC). Fig. 9 .

[0053] Comparative Example 2: Catalytic Activity Test of Free AOX&CAT&GALS

[0054] 100 μL AOX solution (8 mg mL -1 ), 100 μL CAT solution (6 mg mL -1 ), 625 μL GALS solution (4.8 mg mL -1 )、50μL methanol solution (2mol L -1 )、50μL magnesium sulfate solution (200mmol L -1 ) and 50 μL TPP solution were mixed and washed with PBS buffer (50 mmol L -1 , pH 7.5) to 1 mL. Then, the reaction solution was shaken in a 35°C water bath, and 100 μL of the reaction solution was taken out at each time point and 100 μL of sulfuric acid solution (1 mmol L -1 ) to terminate the reaction. The concentration of glycolaldehyde was detected by high performance liquid chromatography (HPLC). Fig. 9 .

[0055] Fig. 9 To compare the catalytic activities of free AOX&CAT&GALS and (AOX&CAT&GALS) / EBCOF@Silica, the conversion of methanol to glycolaldehyde was achieved through the immobilized multi-enzyme catalytic system. Compared with the free multi-enzyme system, the initial cascade reaction rate increased by 143%. The co-immobilization of multiple enzymes in a confined space effectively shortened the enzyme spacing, which was conducive to the rapid transfer of intermediates between enzyme molecules, reduced the diffusion of intermediates to the main solution, and increased the local concentration, thereby accelerating the cascade reaction process.

[0056] In summary, the present invention has developed a method for immobilizing enzymes based on a bifunctional covalent organic framework. EBCOF nanosheets functionalized with quaternary ammonium groups have the dual functions of loading enzymes and inducing silicification. The method first adsorbs the enzyme on the surface of the ionic EBCOF nanosheets through electrostatic interaction, and then the EBCOF nanosheets induce sodium silicate to silicify on its surface to form a silicon oxide coating. The ultrathin EBCOF nanosheets have a high enzyme loading capacity (3.4 mg mg -1 ). At the same time, the formed silica coating effectively protected the enzyme molecules. Compared with the direct adsorption of the enzyme on EBCOF, the presence of the silica coating increased the stability of the immobilized enzyme by 165%, 113%, and 44%, respectively, at pH 11, 50°C, and after 5 cycles. In addition, given the high enzyme loading on EBCOF nanosheets, we extended this method to multi-enzyme catalysis and constructed an immobilized multi-enzyme catalytic system. Compared with the free enzyme system, the initial activity of the immobilized multi-enzyme catalytic system increased by 143%. This method provides a promising method for immobilizing enzymes and expands the application of COF in the field of enzyme catalysis.

[0057] Although the present invention has been described above in conjunction with the accompanying drawings, the present invention is not limited to the above-mentioned specific embodiments, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can make many improvements and changes without departing from the purpose of the present invention, which are all within the protection of the present invention.

Claims

1. An immobilized enzyme catalyst based on a bifunctional covalent organic framework, characterized in that: Covalent organic framework nanosheets are prepared from ethidium bromide and trialdehyde pyrogallol; the obtained covalent organic framework nanosheets have the dual functions of loading enzymes and inducing silicification. The method of immobilizing the enzyme first adsorbs the enzyme on the surface of the ethidium bromide covalent organic framework nanosheet through electrostatic interaction, and then the quaternary ammonium groups on the covalent organic framework nanosheet induce the in-situ formation of a silicon oxide coating on its surface.

2. The method for preparing an immobilized enzyme catalyst based on a bifunctional covalent organic framework according to claim 1, characterized in that: The specific steps are as follows: Step 1) preparing a dispersion of covalent organic framework nanosheets: According to the molar ratio of ethidium bromide to trialdehyde phloroglucinol being 3:2, ethidium bromide and trialdehyde phloroglucinol were dispersed in equal volumes of dimethyl sulfoxide solution, and ultrasonically dispersed uniformly to obtain ethidium bromide dispersion and trialdehyde phloroglucinol dispersion respectively; Add the trialdehyde phloroglucinol dispersion dropwise into the ethidium bromide dispersion, and slowly stir to obtain a mixed solution; The mixed solution was allowed to react at room temperature, diluted with deionized water, and dialyzed in water using a dialysis bag with a molecular weight cutoff of 30,000 Da to obtain a covalent organic framework nanosheet dispersion; Step 2) Preparation of enzyme / covalent organic framework: The covalent organic framework nanosheet dispersion obtained in step 1) is mixed with an enzyme solution, wherein the mass ratio of the enzyme to the covalent organic framework nanosheet is 0.5-20:1; and the mixture is shaken on a shaker at 100-200 rpm for 10-60 min to obtain an enzyme / covalent organic framework dispersion; Step 3) Preparation of immobilized enzyme catalyst based on covalent organic framework: Prepare molar concentration of 10-60mmol L -1 Sodium silicate solution, adjust the solution pH to 7-8 with hydrochloric acid; The sodium silicate solution is added to the enzyme / covalent organic framework dispersion obtained in step 2) at a volume ratio of 1:1, mixed on a shaker, centrifuged to remove the supernatant, and washed with PBS buffer to obtain an enzyme / covalent organic framework immobilized enzyme with a silicon oxide coating grown on the surface.

3. The preparation method according to claim 2, characterized in that: The enzyme is one or more of alcohol dehydrogenase (ADH), alcohol oxidase (AOX), catalase (CAT) and glycolaldehyde synthase (GALS).

4. The preparation method according to claim 2, characterized in that: In step 1), the reaction time at room temperature is 24 hours; the dialysis time is 3 days.

5. The preparation method according to claim 2, characterized in that: In step 3), the shaking speed is 100-200 rpm for 10-60 min; the speed of centrifugal removal of supernatant is 7000-10000 rpm for 2-5 min; the concentration of PBS buffer is 50 mmol L -1 , pH was 7.5, and the number of washings was 3 times.

6. The immobilized enzyme catalyst based on a bifunctional covalent organic framework prepared by any of the preparation methods described in 2-5 is used to achieve the conversion of methanol to ethanolaldehyde.

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