Immobilization Of α-Glucosidase On Gel Beads And Paper And Enzyme Inhibitors Screening

Enzyme is an extremely important catalyst in organisms participating in the whole process of life activities.Thus,when some enzymes in the organism are dysfuncational,it is supposed that a certain disease would occur.Enzyme inhibitors can effectively reduce the activity of disease-related enzymes,thereby achieving disease prevention and treatment.Natural plant drugs are abundant and is an important source for the discovery of new enzyme inhibitor drug molecules.In order to quickly and accurately discover targeted active molecules from complex medicinal plants,immobilized enzyme screening technique based on molecular targeting strategy has been developed rapidly.α-glucosidase,as a target enzyme for diabetes treatment,can hydrolyze carbohydrates in food during digestion to produce glucose and increase blood glucose level.α-glucosidase inhibitors can maintain postprandial blood glucose levels in diabetic patients within a stable range.In this dissertation,α-glucosidase was used as the target enzyme and its immobilization on the carriers,sodium alginate-chitosan gel beads and cellulose filter paper was realized by different methods and were prepared three immobilizedα-glucosidases,and finally enzyme inhibitors were screened from 38 kinds of water extract of traditional Chinese medicine applying the immobilizedα-glucosidases combined with capillary electrophoresis analysis.The main contents are as follows:（1）Preparation of immobilizedα-glucosidase on sodium alginate-chitosan gel beads and enzyme inhibitor screening.Using sodium alginate as the anionic polymer,chitosan as the cationic polymer,and Ca²⁺as the crosslinking agent,the carrier sodium alginate-chitosan gel beads with good biocompatibility,low cost and large surface area were firstly prepared by electrostatic and cross-linking,and the sodium alginate-chitosan gel beads were activated by polyethyleneimine to make their surface positively charged.Then theα-glucosidase was successfully immobilized on the surface of the gel beads by electrostatic adsorption.The experimental results showed that the immobilization method was stable and reliable（RSD=1.9%,n=5）,and the immobilized enzyme not only had good temperature tolerance（70℃）,reusability and storage stability（74%and 57.8%of the initial enzyme activity were retained after 10repeated cycles and 30 days of storage at 4℃,respectively）,but also could be separated from the reaction system instantaneously,which greatly simplified the subsequent analysis.Finally,the immobilizedα-glucosidase was used to screen inhibitors from 12 kinds of water extract of traditional Chinese medicine,among which Sanguisorbae Radix exhibited the strongest inhibitory activity onα-glucosidase.（2）Preparation of immobilizedα-glucosidase on cellulose filter paper coated with chitosan and enzyme inhibitor screening.Cheap and readily available commercial cellulose filter paper was employed as the carrier for enzyme immobilization.Firstly,the surface of cellulose filter paper was coated with chitosan and grafted with the amino groups.And then,polyethylene glycol diglycidyl ether containing two terminal epoxy groups was used as the crosslinking agent,thus theα-glucosidase was successfully immobilized on the surface of cellulose filter paper by ring-opening reaction between the epoxy group and the amino group on the carrier surface and in theα-glucosidase molecule.Compared with the sodium alginate-chitosan gel beads,cellulose filter paper was commercially available and need not to be prepared,thus simplifying the experimental procedure and saving time and cost for enzyme immobilization.The experimental results showed that the immobilized enzyme prepared by this method had better temperature tolerance than the free enzyme,and retained 75%of the initial enzyme activity after 10 repeated uses.Finally,the immobilized enzyme was applied to screen enzyme inhibitors from 14kinds of water extract of traditional Chinese medicines,among which Sanguisorbae Radix,Terminallae Belliricae Fructus,Chebulae Fructus and Phyllanthi Fructus exhibited goodα-glucosidase inhibitory activity.（3）Preparation of immobilizedα-glucosidase on cellulose filter paper coated with a blend of chitosan and polyvinyl alcohol and enzyme inhibitor screening.Aiming at solving the problem of poor mechanical property of cellulose filter paper presented in the previous work,cellulose filter paper was firstly coated with the mixed solution of chitosan and polyvinyl alcohol,thus a structurally stable film was formed on the surface of the carrier through hydrogen bond interaction between chitosan and polyvinyl alcohol molecules thus enhancing the mechanical property of the carrier.Then the surface was further activated with polyethyleneimine to make it positively charged,and finally theα-glucosidase was immobilized on the cellulose filter paper by electrostatic adsorption.The experimental results showed that the tensile strength of the cellulose filter paper modified by chitosan-polyvinyl alcohol blended coating was 1.8 times higher than that of the original one,indicating its mechanical property were obviously enhanced after modification.In addition,compared with the previous one,the immobilized enzyme prepared in this work possessed better storage stability（51.7%initial enzyme activity was retained after 30 days of storage at 4℃）,temperature resistance and alkaline resistance（the enzymatic reaction temperature and p H were 70℃and 8.0,respectively）.Finally,the immobilizedα-glucosidase was used to screen enzyme inhibitors from 12 kinds of water extract of traditional Chinese medicines,among which Sanguisorbae Radix had the strongest inhibitory effect,which was consistent with the previous experimental results.