Cofactor-directed co-immobilization of dual-enzyme on functionalized montmorillonite with enhanced catalytic...

Recently, multi-enzyme cascade catalysis has attracted increasing attention due to the advantages of integrating multiple enzymes, few side reactions and high catalytic efficiency. Herein, a novel dual-enzyme cascade system (GOx-FMt-HRP) was developed through cofactor-directed orientational co-immobilization of glucose oxidase (GOx) and horseradish peroxidase (HRP) onto functional montmorillonite (FMt). The presented method realizes the reconstitution of cofactors and apo-enzymes (enzymes without cofactors), which enables enzymes to be immobilized in specific orientations on the support, thereby effectively reducing changes in their conformation. The results proved that GOx-FMt-HRP exhibited better catalytic stability and reusability than GOx/FMt/HRP (enzymes were co-immobilization on FMt by physical adsorption) and a physical mixture of free enzymes (GOx + HRP). After storage for 1 month, the residual activities of GOx-FMt-HRP, GOx/FMt/HRP, and GOx + HRP were 79.3 %, 60.4 %, and 1.32 %, respectively. Furthermore, GOx-FMt-HRP retained over 74 % activity after 10 uses, which is higher than that of GOx/FMt/HRP (61 %). Moreover, the substrate channeling allowed the prepared GOx-FMt-HRP to show the highest catalytic efficiency for both glucose (2.80 S^-1·mM^-1) and TMB (3.87 S^-1·mM^-1), being approximately 2.6-fold and 2.1-fold higher than that of the GOx + HRP, respectively. This work develops a simple method for dual-enzyme co-immobilization, aiding sustainable enzyme catalysis.