Access to efficient enzymatic channeling is desired for improving all manner of designer biocatalysis. We demonstrate that enzymes constituting a multistep cascade can self-assemble with nanoparticle scaffolds into nanoclusters that access substrate channeling and improve catalytic flux by orders of magnitude. Utilizing saccharification and glycolytic enzymes with quantum dots (QDs) as a model system, nanoclustered-cascades incorporating from 4 to 10 enzymatic steps are prototyped. Along with confirming channeling using classical experiments, its efficiency is enhanced several fold more by optimizing enzymatic stoichiometry with numerical simulations, switching from spherical QDs to 2-D planar nanoplatelets, and by ordering the enzyme assembly. Detailed analyses characterize assembly formation and clarify structure-function properties. For extended cascades with unfavorable kinetics, channeled activity is maintained by splitting at a critical step, purifying end-product from the upstream sub-cascade, and feeding it as a concentrated substrate to the downstream sub-cascade. Generalized applicability is verified by extending to assemblies incorporating other hard and soft nanoparticles. Such self-assembled biocatalytic nanoclusters offer many benefits towards enabling minimalist cell-free synthetic biology.

Cell-free enzymatic cascades have gained increasing interest as one-pot reaction strategies to synthesize complex organic molecules in an efficient, selective, and environmentally sustainable manner. Enzyme immobilization onto nanoparticle surfaces can potentially allow them to benefit from stabilization, localized kinetic enhancement, and to access substrate channeling phenomena in the case of coupled activity. Here, we analyze the activity of benzaldehyde lyase (Bal) when assembled on semiconductor quantum dot (QD). We show that Bal manifests a ∼30% increase in the catalytic rate (k cat) and a greater than 3-fold increase in the enzymatic efficiency (k cat/K M) when displayed on QDs. We then pair Bal with an alcohol dehydrogenase (RADH) within self-assembled QD nanoaggregates to jointly convert benzaldehyde and acetaldehyde to (1R,2R)-1-phenylpropane-1,2-diol, which is a key precursor of the calcium-channel-blocking drug diltiazem. Channeling of the (R)-2-hydroxy-1-phenylpropan-1-one intermediate is confirmed by a ∼50% increase in the coupled enzymatic flux despite the two enzymes displaying a tens of thousands greater difference in the catalytic rates and 3 orders of magnitude difference in their respective Michaelis constants or K M. Bal’s synthetic potential is further highlighted by demonstrating its ability to catalyze product formation with several other structurally diverse aldehyde-displaying substrates. Overall, the results suggest that nanoparticle immobilization has much to offer for augmenting enzymatic biocatalysis in different functional formats.

Most of the complex molecules found in nature still cannot be synthesized by current organic chemistry methods. Given the number of enzymes that exist in nature and the incredible potential...