Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase from Gymnema sylvestre R.Br. Catalyzing Biosynthesis of Steryl Glucosides
Figure 4
Thin layer chromatographic identification of different steryl glycosides (cholesterol 3-O-β-D-glucoside, ergosterol 3-O-β-D-glucoside, and stigmasterol 3-O-β-D-glucoside) formed in the GsSGT catalyzed reaction with UDPG as glucosyl donor and respective sterols as acceptor substrates. (a) Lane 1, ergosterol standard; lane 2 and lane 3, enzyme assay performed with purified GsSGT protein; lane 4, heat inactivated enzyme control; lane 5, substrate (negative) control; lane 6, ergosterol 3-O-β-D-glucoside standard. (b) Lane 1, enzyme assay performed with crude enzyme extract; lane 2, enzyme assay performed with purified GsSGT protein; lane 3, cholesterol standard; lane 4, cholesterol 3-O-β-D-glucoside product standard. (c) Lane 1 and lane 2, GsSGT enzymatic assay (Tris buffer, pH 6.5); lane 3 and lane 4, GsSGT enzymatic assay (Tris buffer, pH 7.5); lane 5, control (Tris buffer + enzyme); lane 6, control 2 (Tris buffer + stigmasterol + protein, no UDP-glucose); lane 7, stigmasterol standard; lane 8, cholesterol 3-O-β-D-glucoside standard.
