Construction And Catalytic Mechanism Of Clay Mineral Based Enzyme Complex Structure Catalytic System

Complex structure enzyme catalytic systems have been widely used in a variety of fields,including biosensing,pharmaceutical synthesis,and bioenergy production due to its advantages of high catalytic efficiency,low byproduct generation,effective reduction of catalytic steps.,et al.A complex structure enzyme catalytic system based on nanomaterials can effectively improve the catalytic stability and reusability of enzymes.However,traditional enzyme immobilization methods usually change the natural molecular environment of natural enzymes,leading to physical and chemical changes in the active center of the enzyme,resulting in a decrease in enzyme stability and activity.Besides,improper supports may reduce the affinity between enzymes and substrates,increase mass transfer resistance between enzymes and substrates,and lead to a decrease in enzyme catalytic efficiency.In addition,improper spatial orientation of enzyme molecules at the support surface interface can disrupt the natural conformation of the enzyme and also lead to a decrease in enzyme catalytic activity.Therefore,how to effectively regulate the catalytic activity of complex structure enzyme catalytic system at the micro level while improving enzyme catalytic stability is still an urgent problem to be solved.Clay minerals are considered ideal support materials for enzyme immobilization due to their natural nanostructures,large specific surface area,easy functionalization and modification,and interfacial reactivity.In addition,the discovery of clay mineral nanozymes provides the possibility of constructing a natural enzyme-clay mineral nanozyme complex structure catalytic reaction system using mineral nanozymes as support.Clay mineral nanozymes possess both the excellent properties of clay minerals and enzymatic catalytic activity.They can not only serve as supports for natural enzymes,effectively improving the stability and reusability of the catalytic reaction system,but also can combine their intrinsic enzymatic catalytic activity with the catalytic properties of natural enzymes to construct complex structure enzyme catalytic system.Therefore,based on the unique structure and catalytic properties of natural clay mineral nanozymes,this dissertation constructs a series of complex structure enzyme catalytic systems using the classic glucose oxidase-peroxidase cascade catalytic reaction system as a model.By regulating the surface properties and microstructure of the support and optimizing enzyme immobilization conditions and methods,the effects of the microstructure and surface properties of the support on the enzyme adsorption kinetics,immobilization efficiency and natural secondary structure of the enzyme were systematically investigated.The effects of surface functionalization and structural modification of supports on catalytic activity and efficiency were investigated.The relationship between the microstructure and catalytic performance of the constructed complex structure enzyme catalytic systems was systematically explored,and its catalytic performance and stability were also evaluated.The results were as follows.Nontronite（NAu-2）possess intrinsic peroxidase-like catalytic activity due to its structure containing a large amount of Fe（III）.The NAu-2 is confirmed to have intrinsic peroxidase-like activity that can catalyze the oxidation of the TMB（colorless）to the ox TMB（blue）in the presence of H₂O₂,providing a theoretical basis for the subsequent construction of a multi-enzyme cascade catalytic reaction system using NAu-2.The physical and chemical properties,surface characteristics,microstructure of support materials,and the binding method between enzymes and supports affect the catalytic performance of multi-enzyme cascade catalytic systems.Inspired by these factors,BSA was used to functionalize NAu-2 by grafting amino and carboxyl groups onto its surface.The modification of NAu-2 by BSA not only improves the stability,water dispersibility,and biocompatibility of NAu-2,but also serves as a stabilizer for natural enzymes,preventing the loss of natural enzyme activity and non-specific adsorption during the immobilization process,which is beneficial for the immobilization and stability of natural enzymes.The results shows that,due to the substrate channelling effect and covalent immobilization method,GOx-BSA/NAu-2 exhibits higher catalytic efficiency and stability than the free enzyme system（GOx+HRP）.The storage stability of GOx-BSA/NAu-2 is higher than that of GOx+HRP.After 20 days of storage,the relative activity of GOx-BSA/NAu-2 was 73.2%,while the GOx+HRP was almost with no activity.In addition,GOx-BSA/NAu-2 maintained a relative activity of approximately 70.5%after 10 cycle used.Traditional enzyme immobilization methods such as physical adsorption and covalent binding generally achieve enzyme immobilization by combining amino acid residues on the enzyme molecular structure with the support,which may cause damage to the natural structure of the enzyme,leading to a decrease in enzyme activity.Enzyme oriented immobilization refers to the orderly fixation of enzymes to specific sites on the support,thereby maximizing the preservation of the natural conformation of the enzyme.In this part,based on prosthetic group affinity,montmorillonite was selected as a support.NH₂-PEG-COOH was used to construct a double arm symmetric I-type structure on the surface of montmorillonite.Subsequently,the cofactors of GOx and HRP were immobilized at both ends of the I-type structure.Finally,the apo-enzyme was immobilized on the support to achieve recombination of the apo-enzyme and the cofactors.The results indicate that the secondary structure change of the GOx-FMt-HRP is smaller than that of the GOx/FMt/HRP,and is closer to the state of natural enzymes.This also indicates that oriented immobilization is beneficial for maintaining the secondary structure of enzymes.Based on the improved catalytic efficiency and excellent catalytic stability of GOx-FMt-HRP,its ability to degrade MB has been further studied investigated.The results shows that the addition of electron mediators can effectively enhance the degradation rate of MB,and the degradation efficiency of GOx-FMt-HRP on MB is about 94.58%within 50 minutes.The radical capture experiments result indicates that the decolorization and degradation of MB mainly due to the oxidation of reactive oxygen species generated by glucose catalyzed by the GOx-FMt-HRP.In summary,this dissertation focuses on“The structure-catalytic activity relationship and the activity micro-regulation mechanism of their enzymatic properties of the complex structure formed by the assembly of enzymes and clay minerals through interfacial interaction”.Based on the interdisciplinary perspective of mineralogy and biochemistry,two complex structure enzyme catalytic systems were designed and constructed,and the surface characteristics,microstructure,the effect and regulation of enzyme immobilization methods on the catalytic performance of cascade catalytic reaction systems reveal the structure-activity relationship between enzyme support microstructure and catalytic activity,and further explore the application prospects of the constructed system in the field of removing water pollutants.This work can provide scientific basis and technical support for the design,targeted screening,and performance optimization of clay mineral based complex structure enzyme catalytic systems,promoting the application of immobilized enzyme technology in the field of environmental remediation.