Bienzymatic nanoreactors composed of chloroperoxidase–glucose oxidase on Au@₃O₄ nanoparticles: Dependence of catalytic performance on the bioarchitecture

Highlights: • Bienzymatic nanoreactor of chloroperoxidase–glucose oxidase on Au@₃O₄ nanoparticles was fabricated. • The nanoreactor integrated two functions: generation of H₂O₂in situ and easy separation using a magnetic field. • The catalytic performance depended on a cage-like structure formed by biotin–glucose Oxidase. • The bienzymatic nanoreactor shows excellent operational stability and reusability. The operational stability of chloroperoxidase (CPO) was considerably enhanced by coupling with glucose oxidase (GOx) because H₂O₂ could be generated in situ from glucose and oxygen. In this paper, a CPO–GOx nanoreactor was fabricated on the surface of Au@₃O₄ nanoparticles through layer-by-layer assembly using the specific avidin–biotin interaction. The X-ray diffraction data indicated the presence of both Fe and Au in the Au@₃O₄ carrier. The Au@₃O₄ displayed a uniform core/shell nanostructure, whereas the nanoparticles of the bienzymatic reactor were larger than the carrier. The catalytic activity of CPO was highly dependent on the structure of the enzymatic nanoreactor. The activity of Au@₃O₄–GOx (inner)–CPO (outer) was 15.5% higher than that of Au@₃O₄–CPO (inner)–GOx (outer). Moreover, Au@₃O₄–GOx–CPO exhibited better thermostability. Au@₃O₄–GOx–CPO retained 53.2% of its initial activity after incubation for 1.0 h at 60 °C and 30.4% of its initial activity after 18 h at 50 °C. Au@₃O₄–GOx–CPO had good reusability. It retained more than 62.4% of its activity after the 12th cycle. This was attributed to the cage-like structure in Au@₃O₄–GOx–CPO, which could effectively prevent the removal of the enzyme molecules from the carrier.