Gold Nanoparticle Templating Increases the Catalytic Rate of an Amylase, Maltase, and Glucokinase Multienzyme Cascade through Substrate Channeling Independent of Surface Curvature

The templating of enzymes has shown myriad advantages, including increased stability and kinetic rates. Specifically, the use of nanoparticles (NPs) as templates has been shown to increase the kinetic rates over larger macroscale scaffolds, in part by overcoming diffusion limitations. Within the field, there is considerable debate over the parameters that optimize this interaction, e.g., NP size, curvature, and surface ligands. Recently much interest has been seen in the pursuit of complete enzyme cascades on nanoscaffolds, with the increased enzyme proximity providing multiple benefits. In this work we demonstrate that multienzyme cascades can be templated on individual gold NPs. Utilizing a three-enzyme cascade of amylase, maltase, and glucokinase, we found a ∼3-fold enhancement in product formation when all three enzymes were bound to the same NP as compared to when the enzymes were bound separately and then combined as well as freely diffusing in solution. This strongly suggests that the increased kinetics was due to substrate channeling. In addition, we investigated the effect of size and curvature on the kinetics of the cascade by using different-shaped gold NPs. Substrate channeling was observed in small and large gold nanospheres (∼30 and 70 nm diameters, respectively) and gold nanostars with an equivalent surface area. Equivalent enhanced kinetics for the enzyme cascade were observed for all of the tested nanoscaffolds, in contrast with what has been reported for single enzymes on gold NPs where changes in curvature resulted in modified kinetics. The results of this work should allow for optimized design of NPs and enzyme cascades for in vitro biocatalysis.