Preparation of immobilized alpha-glucosidase based on magnetic MOF and application of immobilized alpha-glucosidase in enzyme inhibitor screening

Abstract

The invention discloses preparation of immobilized alpha-glucosidase based on a magnetic metal organic framework (MOF) and application of the immobilized alpha-glucosidase in screening of natural enzyme inhibitors. According to the method, ZIF-90 is grafted on the surfaces of Fe3O4 magnetic nanoparticles modified by codeposition of polydopamine and polyethyleneimine, and alpha-glucosidase is synchronously encapsulated in situ in the growth process of the ZIF-90, so that the magnetic MOF immobilized alpha-glucosidase is successfully constructed. On the application level, an in-vitro enzyme inhibition activity evaluation system is constructed, multiple traditional Chinese medicine extracts are selected as research objects, and by means of the characteristic that the magnetic MOF immobilized alpha-glucosidase is easily separated from a reaction system, accurate screening of natural inhibitors or active small molecules is rapidly and efficiently achieved; and a novel screening method is provided for research and development of candidate drugs for diabetes mellitus. The method comprises the following steps: 1, preparing the magnetic MOF immobilized alpha-glucosidase and evaluating the performance of the magnetic MOF immobilized alpha-glucosidase; and 2, the prepared immobilized alpha-glucosidase is used for screening a natural enzyme inhibitor. Based on a magnetic MOF immobilized enzyme technology, the method provided by the invention has the characteristics of rapidness, high efficiency, accuracy, easiness in separation from a reaction system and reutilization of a target receptor alpha-glucosidase, is suitable for screening of alpha-glucosidase inhibitors in complex systems such as traditional Chinese medicines, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to preparation of magnetic MOF-immobilized α-glucosidase and application of magnetic MOF-immobilized α-glucosidase in enzyme inhibitor screening. Background Art

[0002] Enzymes are an important class of biological catalysts in organisms. Many diseases in the human body are closely related to the regulatory effects of key biological enzymes. Enzyme inhibitors can effectively reduce the activity of disease-related enzymes, thereby achieving the purpose of preventing and treating diseases. my country has rich and diverse medicinal plant resources. Their chemical components are characterized by high efficiency, greenness, and low toxicity, and play an important role in the prevention and treatment of diseases. Traditional Chinese medicine has diverse chemical components and complex mechanisms of action, making it an important source of new enzyme inhibitors. However, how to accurately screen out disease-related active ingredients from medicinal plants has become a key issue that needs to be solved urgently. Therefore, it is urgent to develop an efficient enzyme inhibitor screening method targeting disease-related biological enzymes.

[0003] α-glucosidase (GAA) is a carbohydrate hydrolase that plays an important role in the breakdown of sugar metabolism. When polysaccharides in food are initially broken down into oligosaccharides through digestion, GAA specifically recognizes and catalyzes the hydrolysis of the α-1,4-glycosidic bonds at the non-reducing ends of the oligosaccharides, releasing glucose that is absorbed into the blood by small intestinal epithelial cells, leading to increased blood glucose levels after meals. By inhibiting α-glucosidase activity, the release and absorption of glucose can be effectively delayed, thereby achieving stable regulation of blood glucose levels. Therefore, α-glucosidase is considered one of the important drug targets for intervention in diabetes.

[0004] Enzymes, as a class of protein-based biocatalysts, are characterized by high efficiency and specificity. However, their stability and catalytic efficiency are susceptible to environmental factors such as temperature and pH, and they also suffer from drawbacks such as limited reusability and difficulty in separating them from reaction systems. Immobilized enzyme technology can highly concentrate and immobilize free enzymes within a specific space, improving their stability, reusability, and pH and temperature tolerance. In recent years, iron oxide Fe₃O₄ magnetic nanoparticles have been widely used for enzyme immobilization due to their biocompatibility and low toxicity. In particular, the use of magnetic carriers allows for rapid separation of immobilized enzymes from reaction systems, greatly simplifying the process. However, Fe₃O₄ magnetic nanoparticles are prone to aggregation due to dipole forces. Modification with polydopamine and polyethyleneimine can effectively address this issue, improving their dispersibility and stability. Metal-organic frameworks (MOFs) are porous composites composed of metal nodes and organic linkers. They possess advantages such as tunable pore size, high specific surface area, abundant adsorption sites, and good biocompatibility, and have great potential for enzyme immobilization. MOFs are diverse in type, among which ZIF-90, a SOD-like topological structure formed by the reaction of zinc ions and imidazole-2-carboxaldehyde, not only boasts a high specific surface area and uniform pore size, but is also easily modified and synthesized, possessing excellent hydrophilicity and stability, making it an ideal support for enzyme immobilization. However, enzyme immobilization using ZIF-90 requires centrifugation to separate it from the reaction system. Therefore, ZIF-90 was grafted onto Fe3O4 magnetic nanoparticles co-deposited with polydopamine and polyethyleneimine, and α-glucosidase was encapsulated in situ to achieve enzyme immobilization. This approach has been applied to the in vitro screening of enzyme inhibitors, which holds great significance and value for the discovery of new hypoglycemic drug candidates. Summary of the Invention

[0005] The present invention discloses a preparation method of α-glucosidase immobilized on a magnetic metal organic framework (MOF) and its application in enzyme inhibitor screening. Firstly, Fe3O4 magnetic nanoparticles are used as the core magnetic carrier and modified by polydopamine and polyethyleneimine to enhance the dispersibility and stability of the particles; secondly, Zn 2+Based on the coordination self-assembly properties with imidazole-2-carboxaldehyde, a ZIF-90 shielding shell was directionally grown on the modified Fe3O4 surface. At the same time, during the in situ synthesis of ZIF-90, α-glucosidase was simultaneously encapsulated and immobilized to achieve efficient combination of the immobilized enzyme and the magnetic material. Finally, an in vitro enzyme inhibition activity evaluation system was constructed, and a variety of traditional Chinese medicine extracts were selected as research objects. With the help of the easy separation of magnetic MOF-immobilized α-glucosidase from the reaction system, the precise screening of natural enzyme inhibitors was achieved quickly and efficiently. The reliability and practicality of the magnetic MOF-immobilized α-glucosidase for the efficient screening of enzyme inhibitors were systematically verified, providing a new screening method for the development of candidate diabetes drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 Transmission electron microscopy of magnetic Fe3O4 nanoparticles (A) and magnetic MOF-immobilized α-glucosidase (B)

[0007] Figure 2 Hysteresis loop of α-glucosidase immobilized on magnetic MOF

[0008] Figure 3 Reusability of magnetic MOF-immobilized α-glucosidase

[0009] Figure 4 In vitro inhibitory activity of Chinese herbal medicine extracts on magnetic MOF-immobilized α-glucosidase DETAILED DESCRIPTION

[0010] 1. Preparation of magnetic MOF-immobilized α-glucosidase: Dopamine hydrochloride and polyethyleneimine were dissolved in Tris buffer at pH 7.5-10.5, and Fe3O4 magnetic nanoparticles were added. The mixture was ultrasonicated for 10-60 min and shaken in a water bath at 20-50 °C for 2-12 h. After washing with Tris buffer several times, zinc acetate was added to a final concentration of 0.1-1 M. The mixture was shaken in a water bath for 10-60 min, washed with ultrapure water, and then 1-10 U·mL was added. -1 α-glucosidase and 0.1-1 M imidazole-2-carboxaldehyde were shaken in a water bath at 20-50° C. for 1-6 h, 5-10 mL of N,N-dimethylformamide was added, the mixture was allowed to stand, and the magnetic MOF-immobilized α-glucosidase was obtained after washing with ultrapure water.

[0011] 2. Extraction of Chinese medicinal materials: Crush and sieve the Chinese medicinal materials, and use 30-80% methanol-water solution for ultrasonic extraction 2-3 times, each time for 1-3 hours, filter, combine the filtrates, concentrate, and dry to obtain the crude extract of Chinese medicinal materials.

[0012] 3. Screening enzyme inhibitors using immobilized α-glucosidase: The present invention adopts an in vitro α-glucosidase activity inhibition detection method, using a variety of Chinese medicine extracts as test samples and p-nitrophenyl-α-D-pyranoglucoside (p-NPG) as a substrate to evaluate the inhibitory activity of different samples on α-glucosidase, thereby screening for inhibitors with stronger activity. The specific steps are: the crude extract of the Chinese medicine is re-dissolved in PBS buffer containing 10-50% DMSO, pre-incubated with the immobilized enzyme at 0-60°C for 10-60 minutes, then p-NPG is added to initiate the enzymatic reaction, and the reaction system is incubated at 20-70°C for 10-60 minutes. After the reaction is completed, an external magnetic field is used to achieve rapid separation of the immobilized enzyme and the reaction solution. The inhibition rate of different samples on α-glucosidase is calculated by measuring the absorbance value of the supernatant at 405nm, thereby screening for highly active natural inhibitors.

[0013] 4. Advantages of the present invention: 1. The immobilized α-glucosidase prepared by the present invention not only has superparamagnetism and can achieve separation of the immobilized enzyme from the reaction system within 1 minute, but also uses ZIF-90 as the immobilized enzyme carrier. The immobilized enzyme prepared by the biomineralization method has the advantages of good catalytic activity, large enzyme loading capacity, good reusability and strong stability. 2. The prepared immobilized enzyme is used in the screening method for α-glucosidase inhibitors, which has the characteristics of simple operation, rapidity, high efficiency, high throughput, easy separation from the reaction system and repeated use of the target receptor. It is suitable for simultaneous screening of multiple α-glucosidase inhibitors from crude extracts such as medicinal plants. 3. Compared with chemically synthesized inhibitors, natural α-glucosidase inhibitors have the advantages of good efficacy, greenness and low toxicity and side effects.

[0014] The protection scope of the present invention is not limited to the immobilization of α-glucosidase and the screening of Chinese medicine extracts and natural active ingredients. Any technical solutions obtained by technicians through logical analysis, inference and experimentation based on the existing technology in accordance with the ideas of the present invention are within the scope of protection of the claims.

Claims

1. A preparation of α-glucosidase immobilized on a magnetic MOF and its application in enzyme inhibitor screening, characterized in that: The following steps are involved: (1) Preparation of magnetic MOF-immobilized enzyme α-glucosidase; (2) Magnetic MOF-immobilized enzyme α-glucosidase was used for the screening of natural enzyme inhibitors.

2. The preparation of α-glucosidase immobilized on a magnetic MOF and its application in screening natural enzyme inhibitors according to claim 1, characterized in that: In step (1), the preparation of the magnetic MOF-immobilized α-glucosidase specifically includes: dissolving dopamine hydrochloride and polyethyleneimine in tris (hydroxymethylaminomethane) buffer (Tris) to obtain a mixed solution of polydopamine and polyethyleneimine, then adding Fe3O4 magnetic nanoparticles, ultrasonically shaking in a water bath, performing magnetic separation, washing with Tris for several times, and resuspending to obtain PDA / PEI@Fe3O4; adding zinc acetate, shaking in a water bath, washing with ultrapure water for several times, and mixing polydopamine, polyethyleneimine and Zn 2+ The modified Fe3O4 was resuspended in ultrapure water, and α-glucosidase and imidazole-2-carboxaldehyde were added. It was fixed for a period of time and stabilized with N,N-dimethylformamide. After magnetic separation, it was washed with ultrapure water several times to obtain PDA / PEI@Fe3O4@ZIF-90@α-glucosidase, that is, magnetic MOF-immobilized α-glucosidase, which was stored at 4°C for future use.

3. The preparation of α-glucosidase immobilized on a magnetic MOF and its application in screening natural enzyme inhibitors according to claim 1, characterized in that: In step (2), the magnetic MOF-immobilized enzyme is used to screen natural enzyme inhibitors. The specific steps are: using an organic solvent (methanol-water), ultrasonically extracting the traditional Chinese medicine 2 to 3 times, filtering, combining the filtrate, concentrating, and vacuum drying to obtain a crude extract powder. The crude extract powder is redissolved and pre-incubated with the magnetic MOF-immobilized α-glucosidase, and then the substrate p-nitrophenyl-α-D-pyranoglucopyranoside (p-NPG) is added to start the enzymatic reaction. After the reaction is completed, the immobilized enzyme and the reaction solution are quickly separated by an external magnetic field. By measuring the absorbance value of the supernatant at 405 nm, the inhibition rate of different samples on α-glucosidase is calculated, and then high-activity natural inhibitors are screened.

4. The method according to claim 2, wherein The tris (hydroxymethyl)aminomethane buffer (Tris) used has a concentration of 10-100 mM and a pH range of 7.5-10.5; the molar concentration ratio of dopamine hydrochloride to polyethyleneimine is 1:1-1:4; when Fe3O4 is modified using dopamine hydrochloride and polyethyleneimine, the ultrasonic time is 10-60 min, the water bath oscillation temperature is 20-50°C, and the oscillation time is 2-12 h.

5. The method according to claim 2, wherein After adding zinc acetate, the water bath oscillation temperature is 20-50°C for 10-60 min; the α-glucosidase concentration used is 1-10 U·mL -1 , the concentration of imidazole-2-carboxaldehyde is 0.1 to 1 M; the water bath oscillation temperature after adding α-glucosidase and imidazole-2-carboxaldehyde is 20 to 50° C., and the oscillation time is 1 to 6 h.

6. The method according to claim 2, wherein Magnetic MOF-immobilized α-glucosidase has superparamagnetism and can be separated by an external magnetic field within 60 seconds, and can be magnetically separated and recovered in practical applications; the magnetic-based carriers used include but are not limited to Fe3O4, Fe2O3, iron-nickel alloy, manganese-zinc ferrite, nickel-zinc ferrite, magnetic polymer microspheres and other composite magnetic materials; the metal-organic framework complexes used include but are not limited to ZIF-90, ZIF-8, Zn-MOF, Cu-MOF, Fe-MOF, etc.

7. The method according to claim 3, wherein The samples used are traditional Chinese medicine extracts or active ingredients in traditional Chinese medicine; the traditional Chinese medicine extraction solvent is methanol-water (30-80%), and the ultrasonic extraction time is 1-3 hours; in the in vitro magnetic MOF-immobilized α-glucosidase inhibitory activity evaluation model, the preincubation temperature is 0-60°C, the preincubation time is 10-60 minutes, the incubation temperature is 20-70°C, and the incubation time is 10-60 minutes.

8. The method according to claim 1, wherein The disease-modulating key enzymes used include, but are not limited to, α-glucosidase.

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