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 (kcat) and a greater than 3-fold increase in the enzymatic efficiency (kcat/KM) 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 KM. 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.