A versatile colony assay based on NADH fluorescence

Direct visualization of the activity of enzymes expressed by bacterial colonies attached to a solid support, often referred to as “filter assay”, is a powerful strategy for the identification of new or improved biocatalysts. In this work we demonstrate the usefulness of NAD+/NADH coupled enzymatic reactions as visualization tool in such experimental setups. Dehydrogenases, capable of oxidizing or reducing the reaction product released from the bacterial colony were supplemented to the screening solution, together with the screening substrate and a sufficient amount of NAD+ or NADH, respectively. We also examined the screening of directly NAD+/NADH coupled reactions. The release or consumption of NADH in the area of colonies was monitored on behalf of its fluorescence at 450 nm. Excitation was achieved by standard “black-light” UV tubes (340–360 nm). The visible fluorescence signal was recorded using a CCD-camera. We got excellent results for the screening of threonine aldolases and esterases and were able to show the principle utility for amidase, nitrilase, nitrile hydratase, hydroxynitrile lyase and benzaldehyde dehydrogenase active colonies.