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-beta-sophorosyl-19-O-beta-glucosyl-steviol) and rebaudioside-A (tetraglucosylated steviol; 2'-O-beta-glucosyl-13-O-beta-sophorosyl-19-O-beta-glucosyl-stev iol). 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 Km values were 71.4 microM for steviol and 360 microM 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 Km values were 182 microM for steviol, 44 microM for steviolbioside, 95 microM for stevioside, and 385 microM 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 microM, respectively.
Shibata
H
H
Faculty of Agriculture, Shimane University, Japan.
Sawa
Y
Y
Oka
T
T
Sonoke
S
S
Kim
K K
KK
Yoshioka
M
M
D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004224:Diterpenes / Q000302:isolation & purification / Q000378:metabolism*; D045786:Diterpenes, Kaurane; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D018515:Plant Leaves; D010944:Plants / Q000201:enzymology*; D013379:Substrate Specificity; D014462:Ultrafiltration / Q000379:methods; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D004224:Diterpenes; D045786:Diterpenes, Kaurane; D007527:Isoenzymes; C012042:steviol; D005964:Glucosyltransferases; D014532:Uridine Diphosphate Glucose