Preparation And Application Of Immobilized ?-glucosidase On Magnetic Nanoparticles And Paper

Enzymes have been widely used as an efficient and specific biocatalyst in catalysis,sensing,fine chemicals and pharmaceuticals.However,free enzyme exhibits low stability,and is prone to denature and deactivate under extreme environment in practice.In addition,free enzyme is difficult to be recovered and reused.All of these limit its application potential to a certain extent.Immobilization of enzyme is an effective method to improve its operation stability and reusability.The carrier material is an important part of immobilized enzyme,and its structure and property play a key role in the activity and subsequent application of immobilized enzyme.Therefore,developing novel and efficient immobilized carrier materials and immobilized methods is an important issue in the field of enzyme research.In this dissertation,three carrier materials were prepared for immobilizing?-glucosidase,an important therapeutic target enzyme for type 2 diabetes,and the corresponding immobilization methods were developed according to their respective characteristics.The enzymatic reaction kinetics,inhibition kinetics and performance of immobilized enzyme were further studied combined with capillary electrophoresis(CE)analysis.Finally,the immobilized?-glucosidase was applied to screening enzyme inhibitors from traditional Chinese medicine(TCM).The main research contents are summarized as follows:(1)Preparation of immobilized?-glucosidase on magnetic metal organic framework and its application in inhibitor screening.The carboxylated Fe₃O₄ magnetic nanoparticles were prepared by a solvothermal method.Chitosan was deposited on the surface of Fe₃O₄ forming Fe₃O₄@CS core-shell structure through the electrostatic interaction between chitosan(CS)and magnetic nanoparticles and the crosslinking of sodium tripolyphosphate.Then with the addition of Zn²⁺-contained solution,CS chelated with Zn²⁺forming Fe₃O₄@CS@Zn²⁺complex.Finally,with the addition of?-glucosidase and 2-methylimidazole solution,Zn²⁺and dimethylimidazole self-assemble on the surface of magnetic nanoparticles through coordination forming three-dimensional porous framework structure ZIF-8.The morphology,chemical composition,structure and magnetization characteristics of the prepared materials were characterized using transmission electron microscopy(TEM),Fourier transform infrared spectroscopy(FT-IR),X-ray diffraction(XRD)and vibrating sample magnetometer(VSM).The enzymatic reaction kinetics,inhibition kinetics and performance of immobilized enzymes were investigated in combination with CE analysis.The results showed that the immobilized?-glucosidase exhibited higher p H and thermal stability than the free enzyme and retained 73.56%of its initial activity after 10 consecutive catalytic cycles.The immobilized enzyme was further successfully applied to the screening of?-glucosidase inhibitors in methanol extracts of 14 TCMs in combination with capillary electrophoresis analysis,among which Rhei Radix et Rhizoma exhibited the best inhibitory effect.Its inhibitory rate reached 100%at a concentration of 10 mg/m L,and even when the concentration was reduced to 5 mg/m L,the inhibitory rate was still as high as(95.23±0.35)%.(2)Preparation of immobilized?-glucosidase on magnetic covalent organic framework and its application in inhibitor screening.Fe₃O₄ magnetic nanoparticles was employed as the carrier,and organic monomers 1,3,5-tri(4-aminophenyl)benzene and2,5-divinyl-1,4-benzaldehyde were covalentally linked together under the catalysis of acetic acid to form periodic porous skeleton covering the surface of the carrier,thus core-shell type Fe₃O₄@COF was obtained.The as-prepared support material was characterized by TEM,XRD,FT-IR and VSM.?-Glucosidase was further immobilized on Fe₃O₄@COF by covalent coupling with glutaraldehyde as a cross-linking agent.The results showed that the immobilized?-glucosidase exhibited excellent p H and thermal stability,good reusability and storage stability.The prepared immobilized enzymes were successfully applied to the screening of 18 TCMs in combination with CE analysis technique.(3)Preparation of immobilized?-glucosidase on cellulose filter paper and its application in inhibitor screening.Imitating the adhesion behavior of mussels,low-cost catechol and tetraethylene pentamine were co-deposited on the surface of cellulose filter paper(CFP)forming a uniform coating similar to polydopamine,thus CFP was grafted with amino groups.?-Glucosidase was further covalently coupled to the amino-modified CFP via the terminal epoxy groups of polyethylene glycol diglycidyl ether.CFP immobilized?-glucosidase has the feature of instantaneous separation from the reaction medium,which greatly facilitates the subsequent enzymatic reaction kinetics,inhibition kinetics studies,performance evaluation and enzyme inhibitor screening.The results showed that the p H and thermal stability of?-glucosidase were significantly improved after immobilization,and the enzyme activity still retained 68.36%of the initial enzyme activity after repeated use for 10 times.Among the methanol extracts of11 TCMs,Terminalia chebula Retz.,Terminalia billirica and Phyllanthus emblica Linn.showed excellent?-glucosidase inhibitory activity.