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 tent-13-hydroxykaur-16-en-19-oic acid). Its main constituents are stevioside (triglucosylated steviol; 13-O-beta -sophoro syl-19-O-beta-glucosyl-steviol) and rebaudioside-A (tetraglucosylated steviol; 2'-O-beta-glucosyl-13-O-beta-sophorosyl- 19-O-beta-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 M(r) 24,600) catalyzed glucose transfer from UDP-glucose to steviol and steviolmonoside (steviol-13-O-glucopyranoside), but not to other steviol-glycosides. Apparent K-m values were 71.4 mu M for steviol and 360 mu M for UDP-glucose. GTase IIB (subunit M(r) 30,700) showed a broad substrate specificity, acting on steviol, steviolmonoside, steviolbioside (13-O-beta-sophorosyl-steviol), and stevioside. Apparent K-m values were 182 E*M for steviol, 44 mu M for steviolbioside, 95 mu M for stevioside, and 385 mu M 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 K-m values of GTase I and IIB for kaempferol were 12 and 31 mu M, respectively. (C) 1995 Academic Press, Inc.