A magnetic covalent organic framework material immobilized β-d-glucosidase and its application in the conversion of ginsenoside Rb1

Abstract

The application discloses a magnetic covalent organic framework material immobilized beta-D-glucosidase with tetraphenylamino benzene and tetrafluoroterephthaldehyde as monomers and a method for converting ginsenoside Rb1, and comprises the following contents and steps: synthesis of a covalent magnetic organic framework material, immobilization of beta-D-glucosidase, and application of the immobilized enzyme to hydrolytic conversion of ginsenoside Rb1.

Description

Technical Field

[0001] This invention relates to a method for immobilizing β-D-glucosidase using a magnetic covalent organic framework material based on tetraphenylaminobenzene and tetrafluoroterephthalaldehyde as monomers, and its application in the conversion of ginsenoside Rb1, belonging to the field of traditional Chinese medicine biotechnology. Background Technology

[0002] Ginsenoside Rb1 is the main saponin component in ginseng, belonging to the protopanaxadiol type. Under specific conversion conditions, the glycosyl group of ginsenoside Rb1 can be converted into rarer ginsenosides with higher physiological activity, such as ginsenosides Rh2 and Rg3, through hydrolysis. There is a significant demand for rarer ginsenosides with higher physiological activity in scientific research and practical applications.

[0003] Hydrolyzing the glycosyl group of ginsenoside Rb1 can convert it into rare ginsenosides with higher physiological activity. β-D-glucosidase hydrolysis is a commonly used method. However, the enzymes used are mostly free enzymes and cannot be reused, which increases the separation cost. In addition, free enzymes are easily affected by changes in the external environment, leading to a decrease in enzyme activity or even complete loss. Summary of the Invention

[0004] To address the aforementioned drawbacks of conventional methods using free enzymes, this invention provides a method for immobilizing β-D-glucosidase using a magnetic covalent organic framework material based on tetraphenylaminobenzene and tetrafluoroterephthalaldehyde (TEPd). Tetraphenylaminobenzene, TEPd, and amino-based magnetic microspheres are mixed. The tetraphenylaminobenzene and TEPd are then reacted with each other via a Schiff base reaction to prepare the covalent organic framework material. During the reaction, the amino-based magnetic microspheres simultaneously react with the aldehyde groups in the system, resulting in a one-pot preparation of the magnetic covalent organic framework material based on tetraphenylaminobenzene and TEPd. β-D-glucosidase is then chemically bonded to the magnetic covalent organic framework material, thus obtaining β-D-glucosidase immobilized on the magnetic covalent organic framework material.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for immobilizing β-D-glucosidase using a magnetic covalent organic framework material based on tetraphenylaminobenzene and tetrafluoroterephthalaldehyde as monomers and its application in the conversion of ginsenoside Rb1 comprises the synthesis of the magnetic covalent organic framework material, the immobilization of β-D-glucosidase, and the application of the immobilized enzyme in the hydrolytic conversion of ginsenoside Rb1.

[0006] This invention discloses a magnetic covalent organic framework material for immobilizing β-D-glucosidase. The magnetic covalent organic framework material is based on tetraphenylaminobenzene and tetrafluoroterephthalaldehyde as monomers. The immobilization utilizes the adsorption of β-D-glucosidase by the magnetic covalent organic framework material. The magnetic covalent organic framework material is prepared by using tetraphenylaminobenzene and tetrafluoroterephthalaldehyde as monomers, amino magnetic microspheres as magnetic carriers, ionic liquids as solvents, and carboxyl-functionalized ionic liquids as catalysts, via a Schiff base reaction.

[0007] The magnetic covalent organic framework material was synthesized using tetraphenylaminobenzene and tetrafluoroterephthalaldehyde as monomers, amino magnetic microspheres as magnetic carriers, ionic liquids as solvents, and carboxyl-functionalized ionic liquids as catalysts via a Schiff base reaction. The feed ratio of tetraphenylaminobenzene, tetrafluoroterephthalaldehyde, and amino magnetic microspheres was 0.05 mmol:0.10 mmol:20 mg.

[0008] The amino-based magnetic microspheres were synthesized from anhydrous sodium acetate, ferric chloride hexahydrate, ethylene glycol, and hexamethylenediamine, with an optimal ratio of 2g:1g:30ml:5ml, an optimal reaction temperature of 190℃, and an optimal reaction time of 6 hours. After the reaction was complete, the microspheres were washed 3-5 times with water and then 3-5 times with methanol.

[0009] The ionic liquid solvent is one or a mixture of 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, and 1-hexyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide. The volume ratio of the ionic liquid to the molar amount of tetraphenylaminobenzene is 100-1000 µl / 0.05 mmol, with an optimal ratio of 200-400 µl / 0.05 mmol.

[0010] The carboxyl-functionalized ionic liquid catalyst is 1-carboxyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt.

[0011] The reaction temperature of the Schiff base reaction is from room temperature to 100°C, with an optimal temperature of 70°C-85°C, and the reaction time is 12-72 h, with an optimal temperature of 36-48 h.

[0012] The β-D-glucosidase was immobilized by adsorption of β-D-glucosidase onto a covalent organic framework material. The mass ratio of β-D-glucosidase to the covalent organic framework material was 1:3. The immobilization medium was a 0.02M acetate-sodium acetate buffer solution with pH=5.0. The immobilization method was room temperature shaking for 2-6 hours.

[0013] The medium in which the immobilized enzyme is applied to hydrolyze and transform ginsenoside Rb1 is a 0.02M acetate-sodium acetate buffer solution with pH=5.0; the initial concentration of ginsenoside Rb1 is 0.5%-2.0%, with an optimal concentration of 1.0%; the hydrolysis time is 6-24h, and the hydrolysis temperature is 40-60℃, with an optimal concentration of 50℃.

[0014] This invention discloses a method for immobilizing β-D-glucosidase on a magnetic covalent organic framework material based on tetraphenylaminobenzene and tetrafluoroterephthalaldehyde monomers, and its application in the conversion of ginsenoside Rb1. Its key advantage is that immobilizing the enzyme on the magnetic organic framework material carrier results in a more stable enzyme conformation, making it less susceptible to inactivation due to external environmental factors (such as pH, temperature, and inhibitors). The immobilized enzyme exhibits higher operational and storage stability. Furthermore, its magnetic properties facilitate easier recovery and reuse, resulting in low cost and high practicality. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the tetraphenylaminobenzene structure.

[0016] Figure 2 This is a schematic diagram of the structure of tetrafluoroterephthalaldehyde.

[0017] Figure 3 This is a schematic diagram of the reaction between tetraphenylaminobenzene and tetrafluoroterephthalaldehyde.

[0018] Figure 4 The image shows the XRD pattern of amino-magnetic microspheres.

[0019] Figure 5 This is the XRD pattern of a magnetic covalent organic framework material.

[0020] Figure 6 This is a graph showing the determination of ginsenoside Rb1 before the hydrolysis and conversion reaction.

[0021] Figure 7 This is an HPLC chromatogram showing the results after the hydrolysis and conversion of ginsenoside Rb1. Detailed Implementation

[0022] The present invention will now be described in detail with reference to the embodiments and accompanying drawings, but the scope of protection is not limited thereto. Example

[0023] Weigh 6g of anhydrous sodium acetate and 3g of ferric chloride hexahydrate into a 150ml round-bottom flask, add 90ml of ethylene glycol and 15ml of hexamethylenediamine, stir at 55℃ for 2 hours, then transfer to a polytetrafluoroethylene high-pressure reactor and react at 190℃ for 6 hours. After the reaction is complete, wash four times with water and then four times with methanol. Dry under vacuum at 45℃ for 6 hours to obtain amino magnetic microspheres.

[0024] 22.1 mg of tetraphenylaminobenzene, 20.6 mg of tetrafluoroterephthalaldehyde, and 10 mg of amino magnetic microspheres were weighed into a 2 ml stoppered centrifuge tube. 300 µl of a mixture of 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt ionic liquid and 1-carboxy-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt (2:1) was added to ensure uniform dispersion of tetraphenylaminobenzene, tetrafluoroterephthalaldehyde, and amino magnetic microspheres. The reaction was carried out at 80 °C for 24 h. After the reaction was complete, the mixture was washed with methanol until the methanol solution was clarified by adding water. The solid was dried at 80 °C for 12 h to obtain a magnetic covalent organic framework material with tetraphenylaminobenzene and tetrafluoroterephthalaldehyde as monomers.

[0025] Weigh 10 mg of β-D-glucosidase and 20 mg of magnetic covalent organic framework material into a 5 ml centrifuge tube, add 2.0 ml of 0.02 M acetate-sodium acetate buffer solution (pH 5.0), and shake at room temperature for 3 h. Separate magnetically, discard the supernatant, and the solid is the β-D-glucosidase immobilized by the magnetic covalent organic framework material.

[0026] Add 2.0 ml of 0.02 M pH 5.0 acetate-sodium acetate buffer solution and 10 mg of ginsenoside Rb1 to a centrifuge tube, and react with shaking at 50 °C for 6 h. Immobilize β-D-glucosidase with magnetic covalent organic framework material by magnetic separation, and obtain the ginsenoside Rb1 conversion product in the solution.

[0027] Example 2 Weigh 6g of anhydrous sodium acetate and 3g of ferric chloride hexahydrate into a 150ml round-bottom flask, add 90ml of ethylene glycol and 15ml of hexamethylenediamine, stir at 55℃ for 2 hours, then transfer to a polytetrafluoroethylene high-pressure reactor and react at 190℃ for 6 hours. After the reaction is complete, wash four times with water and then four times with methanol. Dry under vacuum at 45℃ for 6 hours to obtain amino magnetic microspheres.

[0028] 22.1 mg of tetraphenylaminobenzene, 20.6 mg of tetrafluoroterephthalaldehyde, and 10 mg of amino magnetic microspheres were weighed into a 2 ml stoppered centrifuge tube. 350 µl of a mixture of 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt ionic liquid and 1-carboxy-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt (2:1) was added to ensure uniform dispersion of tetraphenylaminobenzene, tetrafluoroterephthalaldehyde, and amino magnetic microspheres. The reaction was carried out at 80 °C for 36 h. After the reaction was complete, the mixture was washed with methanol until the methanol solution was clarified by adding water. The solid was dried at 80 °C for 12 h to obtain a magnetic covalent organic framework material with tetraphenylaminobenzene and tetrafluoroterephthalaldehyde as monomers.

[0029] Weigh 10 mg of β-D-glucosidase and 20 mg of magnetic covalent organic framework material into a 5 ml centrifuge tube, add 2.0 ml of 0.02 M acetate-sodium acetate buffer solution (pH 5.0), and shake at room temperature for 3 h. Separate magnetically, discard the supernatant, and the solid is the β-D-glucosidase immobilized by the magnetic covalent organic framework material.

[0030] Add 2.0 ml of 0.02 M pH 5.0 acetate-sodium acetate buffer solution and 15 mg of ginsenoside Rb1 to a centrifuge tube, and react with shaking at 50 °C for 6 h. Immobilize β-D-glucosidase with magnetic covalent organic framework material by magnetic separation, and obtain the ginsenoside Rb1 conversion product in the solution.

[0031] Example 3 Weigh 6g of anhydrous sodium acetate and 3g of ferric chloride hexahydrate into a 150ml round-bottom flask, add 90ml of ethylene glycol and 15ml of hexamethylenediamine, stir at 55℃ for 2 hours, then transfer to a polytetrafluoroethylene high-pressure reactor and react at 190℃ for 6 hours. After the reaction is complete, wash four times with water and then four times with methanol. Dry under vacuum at 45℃ for 6 hours to obtain amino magnetic microspheres.

[0032] 22.1 mg of tetraphenylaminobenzene, 20.6 mg of tetrafluoroterephthalaldehyde, and 10 mg of amino magnetic microspheres were weighed into a 2 ml stoppered centrifuge tube. 300 µl of a mixture of 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt ionic liquid and 1-carboxy-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt (2:1) was added to ensure uniform dispersion of tetraphenylaminobenzene, tetrafluoroterephthalaldehyde, and amino magnetic microspheres. The reaction was carried out at 80 °C for 48 h. After the reaction was complete, the mixture was washed with methanol until the methanol solution was clarified by adding water. The solid was dried at 80 °C for 12 h to obtain a magnetic covalent organic framework material with tetraphenylaminobenzene and tetrafluoroterephthalaldehyde as monomers.

[0033] Weigh 10 mg of β-D-glucosidase and 20 mg of magnetic covalent organic framework material into a 5 ml centrifuge tube, add 2.0 ml of 0.02 M acetate-sodium acetate buffer solution (pH 5.0), and shake at room temperature for 3 h. Separate magnetically, discard the supernatant, and the solid is the β-D-glucosidase immobilized by the magnetic covalent organic framework material.

[0034] Add 2.0 ml of 0.02 M pH 5.0 acetate-sodium acetate buffer solution and 10 mg of ginsenoside Rb1 to a centrifuge tube, and react with shaking at 50 °C for 6 h. Immobilize β-D-glucosidase with magnetic covalent organic framework material by magnetic separation, and obtain the ginsenoside Rb1 conversion product in the solution.

[0035] Figure 6 and Figure 7 This is the XRD pattern of the amino magnetic microspheres obtained in the embodiments of the present invention. Figure 6 and Figure 7 The image shows the XRD pattern of the magnetic organic framework material obtained in Example 1 of this invention. There is a sharp peak in the low-angle region (2θ<10), indicating that a covalent organic framework material has been generated. The other peaks are consistent with the XRD peaks of the amino magnetic microspheres, indicating that the material prepared by this invention is a magnetic covalent organic framework material.

[0036] Figure 6 This is the HPLC chromatogram of the initial reaction of converting ginsenoside Rb1 in Example 3 of the present invention. Figure 7 This is the HPLC chromatogram of the conversion of ginsenoside Rb1 in Example 3 of this invention after 6 hours. Analysis shows that the conversion rate reached over 80% after 6 hours, indicating that the magnetic covalent organic framework material obtained in this invention has good activity for immobilizing β-D-glucosidase. Spectroscopic analysis revealed that the conversion product is ginsenoside Rd; further conversion of ginsenoside Rd is possible with extended conversion time.

[0037] Finally, it should be noted that the embodiments are merely the optimal specific implementations of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A magnetic covalent organic framework material for immobilizing β-D-glucosidase, characterized in that, The magnetic covalent organic framework material is a magnetic covalent organic framework material based on tetraphenylaminobenzene and tetrafluoroterephthalaldehyde as monomers. The immobilization is the immobilization of β-D-glucosidase by utilizing the adsorption of β-D-glucosidase by the magnetic covalent organic framework material.

2. The magnetic covalent organic framework material for immobilizing β-D-glucosidase according to claim 1, characterized in that, The magnetic covalent organic framework material is prepared by using tetraphenylaminobenzene and tetrafluoroterephthalaldehyde as monomers, amino magnetic microspheres as magnetic carriers, ionic liquids as solvents, and carboxyl-functionalized ionic liquids as catalysts, via a Schiff base reaction.

3. The β-D-glucosidase immobilized in the magnetic covalent organic framework material according to claim 2, characterized in that, The feeding ratio of tetraphenylaminobenzene, tetrafluoroterephthalaldehyde, and amino magnetic microspheres was 0.05 mmol:0.10 mmol:20 mg.

4. The β-D-glucosidase immobilized by the magnetic covalent organic framework material according to claim 2, characterized in that, The amino-based magnetic microspheres were synthesized from anhydrous sodium acetate, ferric chloride hexahydrate, ethylene glycol, and hexamethylenediamine, with an optimal ratio of 2g:1g:30ml:5ml, an optimal reaction temperature of 190℃, and an optimal reaction time of 6 hours. After the reaction was complete, the microspheres were washed 3-5 times with water and then 3-5 times with methanol.

5. The β-D-glucosidase immobilized in the magnetic covalent organic framework material according to claim 2, characterized in that, The ionic liquid solvent is one or a mixture of 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, and 1-hexyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide. The volume ratio of the ionic liquid to the molar amount of tetraphenylaminobenzene is 100-1000 µl / 0.05 mmol, with an optimal ratio of 200-400 µl / 0.05 mmol.

6. The β-D-glucosidase immobilized by the magnetic covalent organic framework material according to claim 2, characterized in that, The carboxyl-functionalized ionic liquid catalyst is 1-carboxyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt.

7. The β-D-glucosidase immobilized in the magnetic covalent organic framework material according to claim 2, characterized in that, The reaction temperature of the Schiff base reaction is from room temperature to 100°C, with an optimal temperature of 70°C-85°C, and the reaction time is 12-72 h, with an optimal temperature of 36-48 h.

8. The β-D-glucosidase immobilized in the magnetic covalent organic framework material according to claim 1, characterized in that, The immobilization medium was a 0.02M acetate-sodium acetate buffer solution with pH=5.

0. The immobilization method was room temperature shaking for 2-6 hours. The mass ratio of β-D-glucosidase to covalent organic framework material was 1:

3.

9. The application of β-D-glucosidase immobilized in the magnetic covalent organic framework material according to any one of claims 1-8 in the conversion of ginsenoside Rb1.

10. The application according to claim 8, characterized in that, The medium was a 0.02M acetate-sodium acetate buffer solution with pH=5.0; the initial concentration of ginsenoside Rb1 was 0.5%-2.0%, with an optimal concentration of 1.0%; the hydrolysis time was 6-24h, and the hydrolysis temperature was 40-60℃, with an optimal concentration of 50℃.

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