The Kinetic, Conformational And Electrochemical Studies On Glucose Oxidase

A new method for activity determination of glucose oxidase (GOD) has been explored, and the GODthermal inactivation and conformational lock was analyzed from the point of the thermal inactivation byusing the conformational lock principle, which has important guiding significances for the application ofGOD.In addition, Hydroxylated fullerene (HFs) was used to detect electrochemical behavior of GOD, thusachieving a direct electrochemical on the glassy carbon electrode, and providing a solid foundation for theresearch of new biosensors.Based on the glucose oxidase-horseradish peroxidase-Enzyme coupling system, a new simple, fast andsensitive spectrophotometric method of detecting the glucose oxidase activity was studied by using theexisting laboratory reagents-guaiacol, Research shows that the optimum pH of the new method is5.8,and itdid not make the optimum pH change, in line with the GOD optimum pH,which could accuratelymeasureglucose oxidase activity.Determination of glucose oxidase activity provided by sigma is114.23U/mg, in compliance with sigma activity data(≥100U/mg), proving a accuracy of the new methoddetermination of GOD activity. And new method for determination of enzyme activity was stable andreliable. The standard deviation(RSD) among the enzyme activity data was2.8%, the difference was notsignificant.Adopting the new method for determination of GOD activity by guaiacolto studies the thermalinactivation process and conformational lock of the GOD, GOD is a homodimer widowed protease. Studieshave shown that GOD thermal inactivation process is divided into two linear stages and an inflection point(Optimum temperature, Topt), the data showed that GOD Toptis55℃, when temperature is lower than55℃,the activity change little and stable, when the environment temperature is higher than55℃, will lead to therapid deactivation. According to the theory of the conformational lock,there is a latent period ofinactivation of the enzyme in the process of rapid deactivation of GOD.In which, the GOD present differentforms of intermediates, when reaching a certain time, GOD dimer dissociates into monomer and loses itsinactivation.According to the conformational lock theoretical analysis, research analysis showed that thenumber of the GOD conformational locks is2. Since the discovery of fullerenes was of concern, but its poor solubility in water is not easy to study.However,the fullerene derivatives-hydroxylation of fullerenes (HFs), with better water soluble and superelectrical conductivity, can be applied to the electrochemical study of enzymes.This experiment adopts theHFs modified electrodes to study the GOD electrochemical behavior, The GOD-HFs is strongly modifiedon the surface of glassy carbon (GC) electrode by chitosan (Chi) which has good biocompatibility. Studieshave shown that chitosan membrane provides a good microenvironment for GOD to keep the originalbiological activity, The Cyclic voltammetry of the Chi/GOD-HFs/GC electrode showed that between GODand glassy carbon electrode surface occurred direct,quasi-reversible, The surface diffusion controlledelectron transfer, the potential and electron transfer rate ks are-353±2mV and2.72±0.2s-1,respectively.The electrochemical active substance concentration is8.29×10-11mol/cm; In addition, the results ofChi/GOD-HFs/GC electrode detecting β-D-glucose showed that the linear range of the Chi/GOD-HFs/GCelectrode is5×10-5-1×10-3mol/L. The detection limit is5×10-6mol/L.