Steviol and Steviol-Glycoside: Glucosyltransferase Activities in Stevia rebaudiana Bertoni - Purification and Partial Characterization

The leaves of Stevia rebaudiana Bertoni contain sweet compounds which are glycosides of diterpene derivative steviol (ent-13-hydroxykaur-16-en-19-oic acid). Its main constituents are stevioside (triglucosylated steviol; 13-O-β -sophorosyl-19-O-β-glucosyl-steviol) and rebaudioside-A (tetraglucosylated steviol; 2′-O-β-glucosyl-13-O-β-sophorosyl- 19-O-β-glucosyl-steviol). From the extracts of S. rebaudiana Bertoni, two glucosyltransferases (GTases I and IIB) acting on steviol and steviol-glycosides were isolated, and another distinct activity (GTase IIA) acting on steviol was detected, Purified GTase I (subunit Mr 24,600) catalyzed glucose transfer from UDP-glucose to steviol and steviolmonoside (steviol-13-O-glucopyranoside), but not to other steviol-glycosides. Apparent Km values were 71.4 μM for steviol and 360 μM for UDP-glucose. GTase IIB (subunit Mr 30,700) showed a broad substrate specificity, acting on steviol, steviolmonoside, steviolbioside (13-O-β-sophorosyl-steviol), and stevioside. Apparent Km values were 182 μM for steviol, 44 μM for steviolbioside, 95 μM for stevioside, and 385 μM for UDP-glucose. The two enzymes had a similar optimum pH at 6.5. They also acted effectively on ubiquitous flavonol aglycones, quercetin, and kaempferol and utilized kaempferol at a higher rate than steviol and steviol-glycosides. The apparent Km values of GTase I and IIB for kaempferol were 12 and 31 μM, respectively.