The Bio-mineralization Of Cytochrome C And The Construction And Immobilization Of Multi-enzyme System

enzymes?proteins?are recognized as natural green catalysts with high efficiency and specificity.As an industrial catalyst,it has a wide range of applications in the fields of medicine,fine chemicals,biosensor and environment.Nevertheless,the practical application of enzymes is often limited by the demanding catalytic and storage environment,high cost and difficulty of reuse.At present,based on the excellent properties of different materials,it is a feasible scheme to make full use of the enzyme by fixing the enzyme molecule on the substrate of these materials.In the study of this paper,we chose an efficient and simple bio-mineralization method,and self-assembled bio-composite Cyt c@Cu?Im?₂ obtained by 2-methylimidazole?Im?,CuSO₄ and cytochrome c?Cyt c?to realize the immobilization of Cyt c,and also achieved a significant increase in catalytic reaction rate.On this account,we prepared the bio-composite material*Cyt c@Cu?Im?₂ through the thermal response,and achieved a nearly half reduction in K_m value from21 mM to 10 mM.And thus,the substrate was more easily bound to active center of Cyt c to increase the mass transfer efficiency.Meanwhile,we found that both Cyt c@Cu?Im?₂ and*Cyt c@Cu?Im?₂ showed good stability during long-term storage and incubation of organic solvent ethanol.And the residual activity of them can respectively reach 35%and 18%after 5 times of reuse.In the meantime,GOx&Cyt c@Cu?Im?₂ was also prepared by bio-mineralization to constract multi-enzyme cascade reaction biological composite material by introducing glucose oxidase?GOx?into the highly active Cyt c@Cu?Im?₂ mineralization system,which could not only improve 29 times in K_cat for the catalytic activity of GOx&Cyt c cascade reaction,but also show excellent specificity detection for glucose,and its limit of detection?LOD?for glucose could reach 0.7?M.At the same time,we used GOx&Cyt c@Cu?Im?₂ to detect and analyze the glucose in common carbonated beverages.And glucose content analysis showed that fanta was 2.86 g/100 mL,Sprite was 3.19 g/100 mL and Coca-Cola was 2.33 g/100 mL.In addition,a series of characterization of different bio-mineralized materials was performed,such as SEM and XRD showed that there were significant differences in the microstructure of the materials formed by different enzymes in the same bio-mineralization system.FTIR characteristic peaks were detected for enzymes in materials,and the high weight loss rate of the enzyme-carrying materials in the process of TG,which provided the basis for enzyme immobilization.We also determined the fact that the enzyme was immobilized in the material matrix more intuitively through the fluorescence imaging analysis for these fluorescently labeled enzyme-immobilized materials.