| Enzymes are biological catalysts produced by living cells in the body,which catalyze various biochemical reactions efficiently and play an important role in life activities such as digestion,absorption,respiration,movement and reproduction.α-Glucosidase located in human small intestine is closely related with type 2 diabetes and can hydrolyze polysaccharides in food to produce glucose,thus leading to continuous increase of blood glucose.For diabetic patients,it is difficult to effectively control postprandial blood sugar peak within a safe range.α-Glucosidase inhibitors can inhibit enzyme activity,delay glucose absorption,and achieve the goal of blood sugar control.Plant-derived natural products with diverse structure and rich variety is an important source for the discovery of lead compounds of new drugs.Immobilized enzyme screening technology is to immobilize disease-related enzyme on corresponding carriers by physical or chemical methods and then to be used to screen inhibitors combined with various detection an analysis techniques.The structure and properties of the carrier material are very important for the activity and subsequent application of the immobilized enzyme.Due to its adjustable structure,high mechanical strength and easy separation from the reaction mixture,polymer membrane materials have become an ideal immobilization carrier for enzyme immobilization.In this dissertation,α-glucosidase,as a therapeutic target enzyme for type 2 diabetes,was immobilized on the polymer membrane material by different immobilization methods.Combined with capillary electrophoresis（CE）analysis,the performance,enzyme reaction kinetics and inhibition kinetics of immobilizedα-glucosidase were further investigated.Finally,the immobilizedα-glucosidase was applied to screening enzyme inhibitors from traditional Chinese medicine（TCMs）.The main research contents were summarized as follows:（1）α-Glucosidase immobilization on polymer membrane modified by tannic acid-aminopropyltriethoxysilane and enzyme inhibitors screening.In this work,polyvinylidene fluoride（PVDF）membrane was employed as the carrier forα-glucosidase immobilization.By virtue of the co-polymerization of tannic acid（TA）and 3-aminopropyltriethoxysilane（APTES）and the hydrolysis of APTES,a hierarchical layer-colloidal nanospheres coating was decorated on the surface of PVDF membrane.Subsequently,α-glucosidase was covalently bound to the modified membrane.The morphology and functional groups of the prepared composite were characterized by scanning electron microscopy（SEM）,Fourier transform infrared-Attenuated total reflectance spectroscopy（FTIR-ATR）,and X-ray photoelectron spectroscopy（XPS）.Several parameters affecting the immobilization procedure were investigated thoroughly.The catalytic performance and kinetics behavior of PVDF-immobilizedα-glucosidase were studied.The immobilized enzyme exhibited the enhanced tolerance to temperature and p H value compared with the free enzyme.It possessed good reusability maintaining 77.87%of initial relative activity after 10 recycles.In addition,the immobilizedα-glucosidase could be instantaneously separated from the reaction mixture,thus greatly facilitated the subsequent applications.Finally,combined with CE,α-Glu@TA/APTES@PVDF was utilized to screening potential inhibitors from 13 kinds of TCMs,among which Sanguisorba Radix,Phyllanthus Fructus,Rhei Radix et Rhizoma,Chebulae Fructus and Mori Cortex exhibited positive inhibitory activity.（2）α-Glucosidase immobilization on metal-organic frameworks membrane for enzyme inhibitors screening.Polyvinylidene fluoride（PVDF）membrane was employed as the carrier and modified with dopamine for subsequentα-glucosidase immobilization.First,a polydopamine coating was formed on the surface of the membrane by self-adhensive behavior of dopamine.And then,the rich hydroxyl group of the polydopamine on the membrane was chelated with Zn2+.Finally,with the addition ofα-glucosidase and 2-methylimidazole solution,Zn2+and dimethylimidazole self-assembled on the surface of the coating through coordination interaction forming three-dimensional porous framework structure ZIF-8,and thereby in-situ embeddingα-glucosidase.The as-prepared support material was characterized by SEM,FTIR-ATR,XRD and XPS.The immobilized enzyme showed higher p H tolerance,thermal stability and superior reusability,and maintained 79.32%of its original activity after 10 consecutive catalytic cycles.Combined with CE analysis,α-Glu@ZIF-8@PDA@PVDF was further successfully applied to screening potential inhibitors from 13 kinds of TCMs.The results showed that the Sanguisorba Radix,Spatholobus Suberectus,Chebulae Fructus and Aloe exhibited strongα-glucosidase inhibitory activity.In this dissertation,polymer membrane material was used as carrier forα-glucosidase immobilization by different methods.Compared with free enzyme,immobilized enzyme showed better thermal stability,p H tolerance and reusability.Combined with CE analysis,immobilized enzyme was utilized to screening potential inhibitors from TCMs,many of which exhibited positive inhibitory activity.This study enriched the diversity of membrane materials as immobilized enzyme carriers,and also opened up a new way to search for active ingredients with specific targets from complex systems such as TCMs. |
