D. Hastman, M. Chiriboga and I. Medintz
U.S. Naval Research Laboratory,
Keywords: Biocatalysis, enzyme nanoscaffolds
Summary:At its core, cell-free synthetic biology relies on exploiting enzymatic activity while also seeking to utilize a minimal number of overall components in the most efficient manner possible. This goal requires that the enzymes in a given biocatalytic reaction scheme ideally engage in concerted substrate-product channeling to overcome diffusion limitations. Recent research has revealed that exploiting nanoparticles (NPs) as enzyme nanoscaffolds can both stabilize the enzyme and enhance their localized activity suggesting some new approaches towards achieving high catalytic efficiency. Utilizing the enzymes from glycolysis which serve as the starting point for a number of target biosynthetic products in conjunction with luminescent semiconductor nanocrystals or quantum dots (QDs) and gold nanoparticles (AuNPs) as a prototypical model system, we show that multienzyme cascades can be self-assembled in a facile stochastic to yield QD-enzyme nanoclusters that engage in channeled biocatalysis. The kinetic flux through these nanoclustered systems is increased by several orders of magnitude compared to controls consisting of the same concentration of enzyme alone. Classical enzymatic assays provide strong evidence for a channeling mechanism contributing to the observed enhancements in addition to effects from both the aforementioned stabilization and enhancement of key enzyme components. Iterative optimization of the ratio of each enzyme in these nanoclustered systems utilizing numerical simulations allowed the kinetic flux to be enhanced even further. A variety of other factors such as QD size, relative QD shape (rectangular versus spherical), and enzyme-NP assembly order also influenced the magnitude of catalytic turnover in these self-assembled clustered systems. The complexity achievable to such cascaded systems was further evaluated by increasing the number of participating enzymes from 7 up to a 13 enzyme cascade where (di, poly)saccharide precursor substrates were enzymatically processed all the way to a lactic acid product. The results suggest that the benefits inherent to this self-assembled clustered enzyme approach may be readily transferrable to other multienzyme cascades along with providing a potential plug-and-play type modularity that is not so easily attained in cell-based synthetic biology.