4.15. Glycosyltransferase enzyme assay

Ten μg of recombinant UGT was incubated with 200 μM substrate, 2.5 mM UDP‐glucose, 50 mM Tris pH 7.5 for 3 h at 30°C. Two vols ethyl acetate were added to the reaction, centrifuged for 10 min at 12,000 xg, and the top layer transferred to a new tube. The ethyl acetate extraction was performed once more. The supernatants were pooled and dried under air/nitrogen on an Organomation evaporator and resuspended in methanol.

For β‐glucosidase hydrolysis, rather than resuspend in methanol, the samples were resuspended in 400 μl sodium phosphate buffer (pH 6). The supernatant was divided into two tubes, one as a control without glucosidase digestion, the other for treatment with 6 mg β‐glucosidase for 12 h at 37°C. Following treatment the samples were extracted twice with ethyl acetate, resuspended in 25 μl methanol, and run on the LC–MS as described below.