Organic-inorganic Hybrid Carrier Immobilization Enzyme And Its Application In Inhibitor Screening

The immobilized enzyme technology has been widely used in the field of biological engineering,chemical engineering and medicine,because the temperature and p H tolerance,as well as the reusability of enzyme can be improved after being immobilized.Among them,the use of existing materials to design and develop carriers with excellent performance and low price is an important research direction of enzyme immobilization.Organic-inorganic hybrid carriers combine the advantages of organic and inorganic materials have gradually become one of the ideal carriers of enzyme immobilization.On the other hand,biomimetic mineralization synthesis of organic-inorganic hybrid carriers has many advantages,including mild synthesis conditions,and the formation of inorganic minerals induced by organic components can regulate the morphology and size of inorganic minerals.Therefore,in this study,organic-inorganic hybrid carriers with good specific surface area and morphology was prepared,which used polymer or enzyme as organic components,Ca CO₃ or Cu₃（PO₄）₂·3H₂O as inorganic components,for immobilization of pancreatic kallikrein,lactate dehydrogenase andα-glucosidase,respectively.The prepared materials with immobilized enzymes were used in the study of enzyme kinetics and the evaluation of inhibitor activity.This thesis mainly includes five chapters,including three chapters of experimental research.The first chapter is the literature review.Firstly,the immobilized enzyme,including the immobilization enzyme system,advantages and characteristics,were introduced.Furthermore,several methods of immobilized enzyme were summarized.Then,the immobilized enzyme carriers,including organic carriers,inorganic carriers and organic-inorganic hybrid carriers were described,and biomimetic mineralization and the preparation of organic-inorganic hybrid carriers based on biomimetic mineralization were reviewed.What’s more,the evaluation method of the enzymatic kinetics,inhibition kinetics,reproducibility,reusability and storage stability of immobilized enzymes were summarized.Finally,the purpose,significance and main contents of this study were put forward.In the second chapter,biomimetic mineralization synthesis of poly（sodium4-styrenesulfonate）-mediated Ca CO₃ magnetic microsphere was prepared for pancreatic kallikrein immobilization and was applied for evaluation of enzyme inhibitory activity of natural compounds.Firstly,the poly（sodium 4-styrenesulfonate）-mediated Ca CO₃magnetic microspheres were prepared by a simple spontaneous biomimetic mineralization method to immobilize pancreatic kallikrein,and the structural properties of the materials were characterized by several characterization techniques.Then,the enzymatic reaction conditions were optimized,and the related parameters of the enzyme were determined.Results showed that the immobilized pancreatic kallikrein exhibited optimum p H and temperature at 7.5 and 75°C,respectively.After seven successive cycles,the immobilized pancreatic kallikrein retained 82%of its initial activity.Furthermore,the Michaelis-Menten constant（K_m）and the half-maximal inhibitory concentration(IC₅₀)of serine protease inhibitor（AEBSF）on immobilized pancreatic kallikrein were determined as 3.30 m M and 67.04μM,respectively.Finally,the inhibitory potency of ten natural compounds on immobilized enzyme were evaluated by capillary electrophoresis（CE）analysis.The results showed that berberine,evodine,coptisine and jatrorrhizine had good inhibitory activity on the immobilized kallikrein,and the inhibitory activity with%of inhibition of（43.11±1.74）%,（34.40±1.88）%,（37.98±1.65）%and（28.47±1.04）%,respectively.The binding energy and binding mode of these four active compounds with kallikrein were predicted by molecular docking analysis.In short,biomimetic mineralization is a simple and reliable method for immobilization of enzymes,which can be used for immobilization of other enzymes.The third chapter is biomimetic mineralization synthesis of organic-inorganic hybrid nanoflower for the lactate dehydrogenase（LDH）immobilization and its application in the screening of inhibitors.Firstly,the hybrid nanoflower direct immobilized enzyme was prepared using LDH and Cu₃（PO₄）₂·3H₂O as organic and inorganic components,respectively.The influencing enzymatic reaction factors,enzymatic reaction kinetics and inhibition kinetics parameters were determined after the nanoflower being characterized.The results showed that the optimum p H and temperature of immobilized LDH were 6.5 and 60°C,respectively.After seven successive cycles,the immobilized LDH retained 51%of its initial activity.In addition,the K_m and IC₅₀ of galloflavim on immobilized LDH were determined as 3.05 m M and76.85μM,respectively.Finally,the immobilized LDH was used to evaluate the inhibitory activity of ten natural compounds,among which puerarin apigenoside,aloin A,neochlorogenic acid and icariin showed significant inhibitory activity,and the inhibitory activity with%of inhibition of（53.83±2.82）%,（49.64±1.31）%,（41.42±1.45）%and（34.54±0.47）%,respectively.Finally,the binding energy and binding sites of four active components and LDH were predicted by molecular docking.The fourth chapter is preparation of phytic acid modifiedα-Glucosidase（α-Glu）/Cu₃（PO₄）₂·3H₂O hybrid nanoflower and its application.Based on the study in previous chapter,the specific surface area and stability of the nanoflower were increased by using phytic acid（PA）modification,which improved the stability of the immobilizedα-Glu.The structural properties of the materials were studied by several characterization techniques.Subsequently,the enzymatic reaction conditions such as the p H value and temperature were optimized,and the enzyme kinetics and inhibition parameters were determined.The immobilizedα-Glu had better enzymatic activity under a wide p H range and high temperature than the free one.After seven successive cycles,the PA modifiedα-Glu/Cu₃(PO₄2)·3H₂O hybrid nanoflower could still maintain approximately 63%of its initial immobilized enzyme activity.The K_m and the IC₅₀ of acarbose were determined as 0.77 m M and 15.01μM,respectively.Finally,the material was applied to evaluate the inhibitory activity of ten phenolic compounds onα-Glu.Results indicated that epicatechin gallate（ECG）,gallocatechin gallate（GCG）,epigallocatechin gallate（EGCG）and rosmarinic acid showed good inhibitory activity with%of inhibition of（53.42±2.39）%,（37.28±1.32）%,（37.08±0.63）%and（35.53±0.23）%,respectively.Molecular docking was also used to further analyzed and predict the interaction between the compounds and enzyme.These results indicate that the PA modified hybrid nanoflower is an efficient method ofα-Glu immobilization,which may be applied to immobilize other enzymes in biosensing and biocatalysis.The fifth part is the conclusion and prospective of present study.In this paper,the organic-inorganic hybrid carrier,which combines the biocompatibility of organic material and the excellent mechanical properties of inorganic material,was synthesized based on biomimetic mineralization.It is a simple and stable carrier for immobilization of enzymes.On the other hand,the carriers developed in this study provide more options for the immobilization and applications of enzymes.In addition,the screened inhibitors also provide references for the development of drug for the treatment of related diseases.