(512a) A Specific Non-Covalent Enzyme Immobilization Approach with Nanomaterials to Enable High Activity Retention

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. Here, we want to show an efficient immobilization method based on specific interaction between His-tagged NADH oxidase and functionalized nanomaterials without requiring enzyme purification for immobilization. The first target enzyme was NADH oxidase. 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 (SWCNTs) functionalized with N_α,N_α-bis(carboxymethyl)-L-lysine hydrate. The resulting nanoscale biocatalyst had overcome the foresaid limitations, and demonstrated 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.

Glycerol dehydrogenase (GlyDH) was also immobilized via the same method and the resulting SWCNT-GlyDH could retain 100% free enzyme activity. We further tested this method with other carbon nanomaterials such as multi-walled carbon nanotubes and nanospheres, and the resulting nanomaterial-enzyme conjugates all showed over 80% activity retention.