Self assembling nanoparticle enzyme clusters provide access to substrate channeling in multienzymatic cascades

Abstract

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.

Enzyme assembly on nanoparticle scaffolds enhances cofactor recycling and improves coupled reaction kinetics

“…2C–F. As noted repeatedly, 27,29,33 QD samples lacking GDH present were quite monodisperse in comparison, see ESI Fig. S1 †.…”

“…We next focused on confirming that the GDH tetramer could indeed crosslink with the QDs and form nanoaggregates in a manner similar to that noted previously with several other multimeric enzymes. 27,29,33 Using TEM for visualization and an analytical format that we had implemented previously, 33 the presence of QD aggregates forming was confirmed as shown by the representative TEM micrographs in Fig. 2C–F.…”

“…, dimers or tetramers) and display multiple pendant (His) 6 at their termini, it has been repeatedly confirmed that the QDs and enzymes, whether present individually or as part of a multienzyme cascade, will crosslink into nanoclusters by what appears to be a classical diffusion-limited aggregation (DLA) process. 27–29,33,56 This process also inherently dictates that the final size and structure of the resultant nanoclusters will depend on a complex interplay of the QD and enzyme(s) concentrations present along with their size and shape and the reaction volume amongst other variables. This complex multivariable dependency means that, although the nanoclusters will form, their final size and shape cannot be predicted or controlled.…”

“…Utilizing oxidative glycolysis and select saccharification enzyme(s) as a model system, self-assembled QD cascades capable of improving overall catalytic flux by orders of magnitude were demonstrated. 33…”

“…We then sought to examine this in a coupled reaction utilizing cofactor recycling; we chose lactate dehydrogenase (LDH) as a model due to its well-studied nature and our previous examination of it immobilized onto QDs. 27,33…”