Kinetic Analysis of UGT Enzymes

Positive substrates that were determined via LC-MS were further screened via UDP Glo™ Glycosyltransferase Assay (Promega, Mannheim, Germany) according to the manufacturer’s instruction in order to determine the kinetic parameters of the UGTs. The enzymatic reaction was performed according to [66 (link)] with minor modifications. After starting the reaction by adding UDP-glucose, samples were incubated for 30 min at 30 °C and 400 rpm. The reaction was stopped by adding 12.5 µL 0.6 M HCl and further neutralization with 1 M TRIZMA base. Five µL of the GT reaction was pipetted to a 384 well plate [40 (link)]. The calculation of kinetic data was performed with KaleidaGraph (https://www.synergy.com/; v4.5.4; accessed on 8 September 2021).