Specific and reversible immobilization of NADH oxidase on functionalized carbon nanotubes

Nanotechnology-inspired biocatalyst systems have attracted a lot of attention in enzyme immobilization recently. Theoretically, nanomaterials are ideal supporting materials because they can provide the upper limits on enzyme-efficiency-determining factors such as surface area/volume ratio, enzyme loading capacity and mass transfer resistance. However, common immobilization methods have limited the applicability of these biocatalysts owing to enzyme leaching, 3D structure loss, and strong diffusion resistance. Expensive enzyme purification step is also required for these methods before immobilization. In this work, we show an efficient immobilization method based on specific interaction between His-tagged NADH oxidase and functionalized single-walled carbon nanotubes without requiring enzyme purification for immobilization. We cloned the annotated NADH oxidase gene from Bacillus cereus genome and overexpressed with pET30 vector encoding N-terminal 6× His-tag. The His-tagged NADH oxidase was then immobilized onto single-walled carbon nanotubes functionalized with N(α),N(α)-bis(carboxymethyl)-L-lysine hydrate. The resulting nanoscale biocatalyst has overcome the foresaid limitations, and demonstrates good loading capacity and stability while maintaining 92% maximum activity of the native enzyme. We further demonstrate that the immobilization is reversible and can retain ca. 92% activity for a couple of loading cycles.