Benzaldehyde Lyase Kinetic Improvements, Potential Channeling to Alcohol Dehydrogenase, and Substrate Scope when Immobilized on Semiconductor Quantum Dots

Abstract

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.

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 †.…”

“…, 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.…”

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

“…Our prior efforts looked to understand the catalytic enhancements (e.g., 2–50× improvements in k cat ) observed in enzymes attached to NPs such as semiconductor quantum dots (QDs) or gold NPs (AuNPs) 52 – 54 . Work with amylase, glucokinase, phosphotriesterase, β -galactosidase, benzaldehyde lyase (Bal), alkaline phosphatase, and lactate dehydrogenase (LDH) revealed that enhancements arose from localized nanoenvironment and interfacial effects at the NP-solvent boundary, which served to alleviate key rate-limiting steps (e.g., enzyme-product release or k off ) and also from stabilizing enzyme structures 55 – 61 . Moreover, enhancements were often dependent on NP size, with smaller diameter materials generally favoring catalytic increases 52 .…”

“…Detailed experiments, along with simulations, confirmed intermediate channeling between the QD-colocalized enzymes in the clusters 55 . Moreover, when Bal was recently paired with an alcohol dehydrogenase in similarly cross-linked QD enzyme clusters, their coupled enzymatic flux increased 50% despite the two enzymes differing by >10 4 in catalytic rate and by three orders of magnitude in their respective Michaelis constant, K M 61 .…”

Advances in the One-Step Approach of Polymeric Materials Using Enzymatic Techniques

“…The surface functionalized nanofiber membranes were examined as a support material for enzyme immobilization, which increased the storage stability and reusability, and a novel amylase immobilized poly(vinyl alcohol) (PVA) nanofibers were developed [ 143 ]. Nanoparticle immobilization has contributed greatly to the biocatalysis of enzymes in different functional forms [ 144 ]. In addition to the immobilization of enzymes on substrates by various methods, the self-assembly of enzymes has also been developed, including assembly between different enzymes or with other catalytically active catalysts.…”