Sub-single-turnover quantification of enzyme catalysis at ultrahigh throughput via a versatile NAD(P)H coupled assay in microdroplets

Abstract

AbstractEnzyme engineering and discovery are crucial for a future sustainable bioeconomy, and harvesting new biocatalysts from large libraries through directed evolution or functional metagenomics requires accessible, rapid assays. Ultra-high throughput screening can often require an optical readout, leading to the use of model substrates that may not accurately report on activity for the target reaction and may require bespoke synthesis. In contrast, coupled assays represent a modular ‘plug-and-play’ system, where any pairing of enzyme/substrate may be investigated, if the reaction can produce a common intermediate which links the catalytic reaction to a detection cascade readout. Here we establish a detection cascade, producing a fluorescent readout in response to NAD(P)H via glutathione reductase and a subsequent thiol-mediated uncaging reaction, with a 30 nM detection limit. We demonstrate its utility for the glycosidaseAxyAgu115A (producing monosaccharides from a natural biofuel feedstock) and report a three orders of magnitude improved sensitivity compared to absorbance-based systems, so that less than one catalytic turnover per enzyme molecule expressed from a single cell is detectable. These advantages are brought to bear in plate formats, but also in picoliter emulsion droplets, where enrichments of 950-fold suggest that large libraries can be interrogated against a specific query substrate.