Positional orientating co-immobilization of bienzyme CPO/GOx on mesoporous TiO 2 thin film for efficient cascade reaction.

A multitude of industrial processes are catalyzed by two or more enzymes working together in a cascade way. However, designing efficient enzymatic cascade reactions is still a challenge. In this work, a TiO2 thin film with mesoporous pores was prepared and used as carrier for co-immobilization of chloroperoxidase (CPO) and glucose peroxidase (GOx). By adjusting the dosage of hexadecyltrimethylammonium bromide (CTAB) and the ratio of the two enzymes, CPO and GOx were well distributed and positional orientated to their own appropriate pores to form an ordered "occupation" based on a "feet in right shoes" effect. Moreover, when the pore size was controlled around 12 nm, the enzymes aggregation was inhibited so as to avoid the decrease of activity of enzyme; The catalytic performance of TiO2 -GOx and CPO composites was evaluated by the application of decolorization of Orange G dye in a cascaded manner. The oxidant H2 O2 needed by CPO is generated in situ through glucose oxidation by GOx. Upon co-immobilization of CPO and GOx on the same carrier, a large increase in the initial catalytic efficiency was detected when compared to an equimolar mixture of the free enzymes, which was four times greater. Moreover, the affinity of the enzyme toward substrate binding was improved according to the kinetic assay. The thermal stability of TiO2 -GOx and CPO composites were greatly improved than free enzymes. The TiO2 -GOx and CPO composites can be easily separated from the reaction media which facilitate its recycle use.