Preparation Of Immobilized Glucose Oxidase Based On Metal-Organic Frameworks(ZIF-7)

Glucose oxidase（Glucoseoxidase,GOx）has attracted wide attention in the fields of industrial production,biomedicine and biosensor because of its safety,high efficiency,mild reaction conditions and high specificity.However,the essence of GOx is protein,which has poor stability and easy to be inactivated in adverse environment,which limits its industrial application.The results showed that the immobilized enzymes were able to improve the stability and reusability of the enzymes and reduce the production cost.However,there are some problems in the traditional immobilization technology and carrier,such as inactivation of enzymes,stability of immobilized enzymes and complex preparation process.In order to solve these problems,a new method of immobilization of GOx in ZIF-7（Zeolitic Imidazolate Frameworks-7）by in-situ embedding method in aqueous phase was proposed for the first time.Then,GOx-ZIF-7 biocomposites with polydopamine（PDA）modified layer were synthesized,and their properties were studied.In addition,the mechanism of GOx in-situ embedded in ZIF-7 was preliminarily studied by molecular docking technology and modern spectral technology,which provided a theoretical basis for a comprehensive and in-depth understanding of the preparation of enzyme immobilized by in-situ embedding method.The main contents of this thesis are as follows:1.In this paper,the biocomposite GOx@ZIF-7 was synthesized by in-situ embedding method in aqueous phase for the first time.Then,the immobilized enzyme GOx@ZIF-7/PDA was prepared by self-polymerization of dopamine.The preparation conditions were optimized by single factor experiment.The optimum preparation conditions were as follows:p H of enzyme solution was 7.0,enzyme concentration was 5 mg/m L,concentration of Benzimidazole（Bim）was 50 m M,molar ratio of Zn²⁺to Bim was 1:9,immobilization temperature was 35℃,immobilization time was 40 min,and dopamine concentration was 2 mg/m L.The activity recovery of GOx@ZIF-7/PDA was 43.34±2.00%.The GOx@ZIF-7/PDA was characterized by scanning electron microscopy,infrared spectroscopy and powder X-ray powder diffraction.Compared with free enzyme,the immobilized enzyme GOx@ZIF-7/PDA has significantly improved thermal stability,p H stability,storage stability,and reusability,which is expected to achieve low-cost long-term stable application of enzyme catalysis.2.The interaction between GOx and ZIF-7 precursors（zinc ion and benzimidazole）was studied by molecular docking and modern spectroscopic techniques.The results showed that different solution temperature and p H value of buffer leaded to the conformational change of GOx.Zinc ion and benzimidazole had multiple binding sites on the enzyme,thus inducing the accelerated synthesis of ZIF-7 around the enzyme.In addition,in the process of binding to ZIF-7 precursor,the structure of the enzyme changed,but the changes had almost no effect on the activity of the enzyme.3.In order to further improve the activity recovery of the immobilized enzyme,the surface of GOx was modified by double-ended Dopamine grafted polyethylene glycol（Dopamine-PEG-Dopamine,DA-PEG-DA）,and then the biocomposite DA-PEG-DA/GOx@a ZIF-7/PDA was prepared by in-situ embedding method and self-polymerization of dopamine in aqueous phase.The activity recovery of DA-PEG-DA/GOx@a ZIF-7/PDA was 99.02±1.03%.Compared with the free enzyme,DA-PEG-DA/GOx@a ZIF-7/PDA has higher substrate affinity,stability and reusability.DA-PEG-DA/GOx@a ZIF-7/PDA was characterized by transmission electron microscope,X-ray photoelectron spectroscopy,infrared spectroscopy and so on.To detect glucose,it was found that DA-PEG-DA/GOx@a ZIF-7/PDA had good substrate selectivity and wider detection range.This study not only provides a preparation method of immobilized enzyme with high activity,high stability and low enzyme leakage rate for glucose detection,but also provides a theoretical basis for a more comprehensive and in-depth understanding of the preparation process of immobilized enzyme by in-situ embedding method.