The development of new methods for fabricating artificial multienzyme systems has attracted much interest because of the potential applications and the urgent need for multienzyme catalysts. Controlling the enzyme ratio is critical for improving the cooperative enzymatic activity in multienzyme systems. Herein, we introduce a versatile strategy for fabricating a multienzyme system by coimmobilizing horseradish peroxidase (HRP) and glucose oxidase (GOx) on magnetic nanoparticles multifunctionalized with dopamine derivatives through DNA-directed immobilization. This multienzyme system exhibited precise enzyme ratio control, high catalytic efficiency, magnetic retrievability, and enhanced stability. The enzyme ratio was conveniently adjusted, as required, by regulating the quantity of functional groups on the multifunctionalized nanoparticles. The optimal mole ratio of GOx/HRP was 2:1. The Michaelis constant Km and specificity constant (kcat/Km, where kcat is the catalytic rate constant) of the multienzyme system were 1.41 mM and 5.02 s-1 mM-1, respectively, which were approximately twice the corresponding values of free GOx&HRP. The increased bioactivity of the multienzyme system was ascribed to the colocalization of the involved enzymes and the promotion of DNA-directed immobilization. Given the wide variety of possible enzyme associations and the high efficiency of this strategy, we believe that this work provides a new route for the fabrication of artificial multienzyme systems and can be extended for a wide range of applications in diagnosis, biomedical devices, and biotechnology.
Keywords: DNA-directed immobilization; enzyme coimmobilization; multienzyme system; multifunctionalized magnetic nanoparticles; ratio control.