Fluorophoric Assay for the High-Throughput Determination of Amidase Activity

An assay has been developed for the high-throughput identification of amidase activity. Amines released from the enzyme-catalyzed hydrolysis of corresponding amides were detected by the formation of a fluorescent dye by coupling with 4-nitro-7-chloro-benzo-2-oxa-1,3-diazole (NBD-CI). Using this format, 22 lipases and esterases were tested for their ability to hydrolyze aromatic substituted N-acylamines in a microtiter plate format. Identified active enzymes were further characterized toward a broad range of compounds to determine the influence of substrate structure on activity. For recombinantly produced esterases, it could be shown that the assay works with high reproducibility and sensitivity, even in the presence of amino acids and proteins present in culture media and cell debris.