Oriented Co-immobilization Of Multi-enzyme Systems Based On Prosthetic Group Affinity

As the multi-enzymatic cascade broadly applied, co-immobilization of multi-enzyme systems becomes increasingly attractive. It can fully fuse the catalytic properties of different enzymes, and promote the concentration of intermediate products in motion, the application range will be expanded and the whole reaction efficiency of multi-enzymes may be improved. Consequently, it can overcome the limits that a single enzyme reaction system only can express a simplex catalysis to a large extent. The enzymes are fixed to carriers with the sequence by oriented immobilization, which can keep the natural conformation of enzymes and be conductive to preserve catalytic function.Both of the catalytic active center of glucose oxidase(GOD) and cholesterol oxidase(ChOx) are flavine adenine dinucleotide(FAD). Glucose and cholesterol can be catalyzed separately by GOD and ChOx, generating corresponding products and H2O2. The enzymes will be injured by H2O2, and the reactions might be inhibited by H2O2 as intermediate product. Horseradish peroxidase(HRP) has a hemin active center, and it can consume H2O2 and facilitate forward reaction ultimately. Therefore, GOD and ChOx are severally able to be coupled with HRP to fabricate multi-enzyme cascade. The biological affinity between prothetic group and apo-protein can be utilized to co-immobilize these three category oxido-reductase. This method can realize high degree orientation of multi-enzyme without chemical modification of enzymes.In this work, the multi-enzymatic cascade system was prepared through the oriented immobilization of prothetic group. Firstly, polyaniline(PANI) was used to establish asymmetric Y! bridge. After that, FAD and Hemin were immobilized to the functional carrier. Finally, the apo-enzymes were reconstituted with cofactors, and both of the GOD-HRP system and ChOx-HRP system were build.The study showed that the electrochemical activity of the GOD-HRP system increased nearly 3.6 times than that of the single GOD, and the linear range of substrate reaction was 3~50mM. In addition, the electrochemical activity of the ChOx-HRP system increased nearly 2.1 times than that of the single ChOx, and the linear range of substrate reaction reached 3~80mM. The thermal stability and acid-base resistance properties of oriented co-immobilization multienzyme system are also enhanced. The GOD-HRP system can retain upper catalytic activity in pH 6.5-7.0, and optimal reaction temperature was 35 T. And the ChOx-HRP system can keep upper catalytic activity in pH 6.0-7.5, and applicable temperature was 35-45 T.The length of conductive polymer could influence the electrochemical activity of multi-enzyme systems. On account of surface group, deficiency and conjugated structure of graphene oxide(GO) and PANI, the transportation of current was intensively impacted. The electrochemical activity was gradually enhanced with the improvement of the reduction degree of support.