A highly specific glucosyltransferase is involved in the synthesis of crocetin glucosylesters in Crocus sativus cultured cells

Saffron (Crocus sativus) stigmata contain rare water-soluble carotenoids an d the major one is crocin; the crocetin digentiobiosyl-ester. Previous stud ies indicated that two glucosyltransferases mig ht be involved in the form ation of crocetin glucosyl- and gentiobiosyl-esters (Dufresne et al. 1997). A UDP-Glc:crocetin 8,8'-glucosyltransferase involved in the biosynthesis o f crocetin monoglucosyl- and diglucosyl-esters was extracted from saffron c ell cultures and purified 300-fold by gel filtration chromatography and pre parative IEF electrophoresis, with a recovery of 13%. The purified enzyme p reparation was highly specific for crocetin and formed ester bonds between the glucose moiety of UDP-Glc and the free carboxyl functions of crocetin. The enzyme did not add other glucose units to the glucosyl-esters to form c rocetin gentiobiosyl-esters. A crude desalted extract of the same material was less specific and formed glucosyl-esters with several other compounds, including abscisic and retinoic acids. The purified preparation was active between pH 4.4 and 4.6. SDS-PAGE revealed a major band at 26 kDa while the native molecular mass determined by gel filtration was in the range of 49 t o 55 kDa. The study provides concrete evidence for the hypothesis that more than one glucosyltransferase is involved in the biosynthesis of crocetin g lycosyl-esters in saffron.