Preparation Of Immobilized Enzyme Based On Dopamine Coating Strategy And Enzyme Inhibitors Screening

Enzymes are the boosting agents of various chemical reactions in organisms,which are closely related to the metabolic process of human body.If abnormal conditions such as impaired or defective enzyme functions occur in the body,diseases will occur.In modern medicine,enzymes are not only an important indicator of disease diagnosis,but also the main target of drug discovery.α-glucosidase in human small intestine can hydrolyze disaccharides in food to produce glucose,which leads to continuous increase of glucose absorbed into blood.For diabetic patients,it is difficult to effectively control postprandial blood glucose peak in a safe range.α-glucosidase inhibitor is considered to be effective in inhibiting enzyme activity,delaying the absorption of sugar into blood after meals,and achieving the goal of controlling blood sugar.Natural products are the main source for the discovery of new drug lead compounds.Plants are rich in species and contain complex chemical components,which contain potentially effective enzyme inhibitors.Traditional screening methods are heavy in workload,long in cycle and easy to produce false positive or false negative results.Immobilized enzyme technology has developed rapidly in recent years and has been widely used in the activity screening of natural products.In this paper,α-glucosidase was selected as the target enzyme,immobilized enzyme was prepared based on dopamine surface coating strategy,and combined with capillary electrophoresis analysis technology,the enzyme inhibitory activity of traditional Chinese medicine extracts was evaluated accurately.The main research contents include:(1)Preparation of α-glucosidase immobilized on cellulose filter paper coated with polydopamine and screening of enzyme inhibitor.Using cheap and effective cellulose filter paper as immobilized carrier,a poly-dopamine functional coating was produced on the surface of filter paper by the self-polymerization adhesion behavior of dopamine,and then α-glucosidase was covalently bonded to the modified filter paper by Schiff base reaction and Michael addition reaction.Compared with freeα-glucosidase,α-glucosidase immobilized on filter paper has better operation stability and reusability,and its instant separation from enzyme reaction solution can greatly simplify the subsequent operation steps.Taking acarbose as a model inhibitor,the reliability of the filter paper immobilized enzyme in the application of enzyme inhibitor screening was demonstrated by verifying the inhibition kinetics of the immobilized enzyme.Finally,α-glucosidase immobilized on polydopamine modified filter paper was applied to screen enzyme inhibitors of 11 kinds of traditional Chinese medicine extracts.(2)Preparation of α-glucosidase immobilized on dopamine-polyethyleneimine co-deposited cellulose filter paper and screening of enzyme inhibitors.On the basis of the previous work,the surface of filter paper was modified by polyethyleneimine-assisted dopamine co-deposition,which made the surface of filter paper more uniform and smooth.Polyethylenimine gave high density positive charges to the surface of filter paper,and α-glucosidase could be immobilized on coated filter paper by electrostatic adsorption.The immobilization process was fast and mild conditions did not significantly change the properties of enzyme.The results showed that the immobilized α-glucosidase had excellent reusability and good reproducibility.In addition,taking acarbose as a model inhibitor,the application reliability of this filter paper immobilized enzyme in the screening of enzyme inhibitors was verified by the inhibition kinetics of immobilized enzyme.Finally,combined with capillary electrophoresis,immobilized α-glucosidase was used to screen enzyme inhibitors for10 kinds of traditional Chinese medicines.(3)Preparation of α-glucosidase immobilized on dopamine-polyethyleneimine modified capillary column and on-line enzyme inhibitor screening.The previous work proved that dopamine-polyethyleneimine co-deposition strategy is a simple and effective surface modification method,and endows the carrier surface with the ability of enzyme immobilization.In this work,the inner wall of the inlet end of capillary column was modified by dopamine-polyethyleneimine co-deposition,and then α-glucosidase was combined with the inner wall of capillary column by electrostatic adsorption.By injecting the substrate solution containing(or not containing)the sample to be screened,the enzyme catalytic activity and the inhibition activity of the sample were determined on line.The results show that the capillary column enzyme microreactor has excellent reusability and reproducibility.The verification of acarbose model inhibitor shows that the established online screening method is reliable and effective.Finally,the capillary column enzyme microreactor was applied to screen α-glucosidase inhibitors from 20 traditional Tibetan medicine extract.