Site-specific And Quantitative Co-localization Of Double Enzyme System

Compared with the free enzyme,the immobilized enzyme has a series of advantages such as high storage stability,strong vigor,and easy recovery.Therefore,people have studied the immobilization of enzymes long time ago.Initially,the immobilization of single enzymes began.Later,people began to study the immobilization of multi-enzyme systems,but the initial immobilization of multi-enzyme systems was relatively crude.In order to improve the synergistic effect of the enzyme and improve the catalytic efficiency of the multi-enzyme system,site-specific and quantitative co-localization of multi-enzyme system becomes the focus of research today.This topic provides a new scheme for the site-specific and quantitative co-localization of double enzymes system.First of all,we must select a suitable immobilization carrier to fix the double enzyme system.In the present study,magnetic nanoparticles of Fe3O4 served as solid supports mainly due to their unique properties such as superparamagnetism,high surface area,large surface to volume ratio,and easy separation under external magnetic fields.Then,about the immobilization method.We chose to use the specific binding property of anchoring protein and adhesion protein to link the double enzyme system we need to immobilize on the anchoring protein,and then attach the scaffold protein to the magnetic nanoparticles of Fe3O4,which not only achieves specificity,and the amount of binding can be controlled by controlling the amount of binding domain.Thereby realizing the sequencing and quantitative fixation of the double enzyme system.Finally,the choice of a double enzyme system.We chose glucose oxidase and a catalytically active oligonucleotide chain G-quadruplex,not only because the catalytic reaction of these two enzymes can be judged by simple color development,but also the G-quadruplex is a single strand of nucleic acid can be simply extended and linked to glucose oxidase,which not only greatly shortens the mass transfer distance of the cascade reaction,but also prevents the two enzymes from interacting with each other due to too close distance.In this study,the structural specificity of scaffold proteins is used to co-immobilize glucose oxidase and G-quadruplex double enzyme system.The final activity of the immobilized enzyme was recorded 3838 U/g Fe3O4 magnetic nanoparticles.