High Activity and Convenient Ratio Control: DNA-Directed Coimmobilization of Multiple Enzymes on Multifunctionalized Magnetic Nanoparticles

Abstract

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 K and specificity constant (k/K, where k is the catalytic rate constant) of the multienzyme system were 1.41 mM and 5.02 s mM, 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.

Hierarchical Micro‐ and Mesoporous Zn‐Based Metal–Organic Frameworks Templated by Hydrogels: Their Use for Enzyme Immobilization and Catalysis of Knoevenagel Reaction

“…These values verified the remarkably enhanced enzyme catalytic efficiency enabled by the large pore sizes of the support that captured more substrates and facilitated the diffusion of both substrate and product molecules. Similar findings have also been discovered by other researchers . For example, Yang et al co‐immobilized GOx and HRP at the surface of magnetic nanoparticles by DNA‐directed immobilization, and its k cat / K m value was 11.8‐fold better than that of the free enzymes .…”

“…Similar findings have also been discovered by other researchers. [64][65][66][67] For example, Yang et al co-immobilized GOx and HRP at the surface of magnetic nanoparticles by DNA-directed immobilization, and its k cat /K m value was 11.8-fold better than that of the free enzymes. [66] In another work reported by the same group, the k cat /K m value of immobilized enzymes was approximately twice of free GOx&HRP.…”

Controllable and high‐performance immobilized enzyme reactor: DNA‐directed immobilization of multienzyme in polyamidoamine dendrimer‐functionalized capillaries

“…For the specific steps used to prepare the enzyme–DNA conjugates and fluorescence‐labeled enzymes, please refer to our previous work . In the present study, the enzyme–DNA conjugates were α′‐GOx and β′‐HRP (strands α′ and β′ in Supporting Information Table ), and the fluorescence‐labeled enzymes were FITC‐labeled HRP and RhB‐labeled GOx.…”

“…We used various concentrations of glucose solution, while keeping the concentration of the other substrate (ABTS) constant. The experiment was carried out under the optimized enzymatic conditions, and the enzymatic temperature was maintained at 37°C, in accordance with our previous work [39][40][41]. As shown in Fig.…”

“…The technique provides a biocompatible, mild, and efficient method of immobilizing specific enzymes . Moreover, the use of DNA double‐strand hybridization and dehybridization can achieve reversible immobilization of the enzymes, while reducing the cost and enhancing the efficiency of enzyme immobilization . However, maintaining the catalytic activity and stability of the immobilized enzymes does not fully meet the current need for open‐tubular CE‐integrated IMER, and the limited enzyme loading capacity is often problematic.…”