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. 2016 Jun 2;212(1):123–135. doi: 10.1111/nph.14039

Figure 3.

Figure 3

In vitro enzyme assays for determining activity of Glycyrrhiza uralensis UDP‐dependent glucuronosyltransferase (GuUGAT). (a) Sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS‐PAGE) electropherogram of proteins expressed in Escherichia coli. M, standard protein markers; 1, crude protein after isopropyl 1‐β‐D‐thiogalactoside (IPTG) induction; 2, purified GuUGAT protein; 3, concentrated GuUGAT protein. (b) Continuous two‐step glycosylation reaction catalysed by GuUGAT. (c) Chemical reference substance determined by high performance liquid chromatography‐linear ion trap‐orbitrap mass spectrometry. 1, glycyrrhetinic acid (retention time: 5.84 min); 2, glucopyranosiduronic acid (retention time: 5.18 min); 3, glycyrrhizin (retention time: 4.46 min); MS fragments of glycyrrhizin are shown subsequently. (d, e) For each enzyme assay, (d) 50 μM glycyrrhetinic acid or (e) 50 μM glucopyranosiduronic acid was used as a substrate. Extracted chromatographic peaks for the substrates used are highlighted with asterisks (*). Enzymatic products are as indicated: 1, glycyrrhetinic acid (retention time: 5.83 min); 2, glucopyranosiduronic acid (retention time: 5.18 min); 3, glycyrrhizin (retention time: 4.48 min). MS fragments of peak 3 are shown subsequently.