Optimizing the conversion of phosphoenolpyruvate to lactate by enzymatic channeling with mixed nanoparticle display

Co-assembling enzymes with nanoparticles (NPs) into nanoclusters allows them to access channeling, a highly efficient form of multienzyme catalysis. Using pyruvate kinase (PykA) and lactate dehydrogenase (LDH) to convert phosphoenolpyruvic acid to lactic acid with semiconductor quantum dots (QDs) confirms how enzyme cluster formation dictates the rate of coupled catalytic flux (kflux) across a series of differentially sized/shaped QDs and 2D nanoplatelets (NPLs). Enzyme kinetics and coupled flux were used to demonstrate that by mixing different NP systems into clusters, a >10× improvement in kflux is observed relative to free enzymes, which is also ≥2× greater than enhancement on individual NPs. Cluster formation was characterized with gel electrophoresis and transmission electron microscopy (TEM) imaging. The generalizability of this mixed-NP approach to improving flux is confirmed by application to a seven-enzyme system. This represents a powerful approach for accessing channeling with almost any choice of enzymes constituting a multienzyme cascade. [Display omitted] •Assembly of enzyme nanoclusters with mixed nanoparticle types increases enzymatic channeling•Mixing nanoparticle sizes and shapes enables structural optimization of nanoclusters•Designer cascades with almost any enzyme type should be possible with this approach•A mixed nanoparticle approach is demonstrated with two- and seven-enzyme systems The ability to synthesize complex molecules in a green manner using enzymes represents a sustainable strategy toward replacing many fossil fuel-derived chemical feedstock and energy-intensive synthetic processes. To offer greater synthetic versatility, such synthetic biology approaches must expand beyond the limits of cell-based systems and bypass cellular toxicity to create new molecules that cells cannot make. Thus, methods to join multienzyme cascades together in vitro to function in the most efficient manner possible are highly desirable. We show how different nanoparticle materials allow display and clustering of two coupled enzymes into aggregates that access channeling phenomena and increase catalytic flux by orders of magnitude. Mixing different nanoparticles together in this system enhances reactions beyond the capabilities of a single-size nanoparticle material and enables for selection of a nanoparticle-enzyme cluster that has been more structurally optimized for increased product yield with fewer reactants. Enzymatic channeling is a highly ef