Montri ChulavatnatolBhinyo PanijpanJisnuson SvastiPanpaka Chalermisrachai2023-10-182023-10-18198819882023Thesis (M.Sc. (Biochemistry))--Mahidol University, 1988https://repository.li.mahidol.ac.th/handle/20.500.14594/90496A glucosyltransferase which catalyzes the conjugation of ace-tone cyanohydrin to glucose in the last step of linamarin biosynthesis was isolated from young leaves of cassava (Manihot esculenta Crantz.). The glucosyltransferase was found to be a soluble enzyme and was partially purified by 40-80% ammonium sulphate fractionation followed by Sephadex G-200 column and Mono Q HR 5/5 column chromatography. These steps resulted in a purification of 6 folds. The partially purified glucosyltransferase possessed negligible linamarase and little UDP-glucose hydrolysis activity. The native molecular weight of cassava glucosyltransferase was 46,000. The partially purified preparation consisted of five major bands of 13,000; 21,000; 25,000; 31,000 and 39,000 separated by polyaccrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The enzyme activity was unstable but could be kept at -20 degree C for 17 days with 50% loss of the activity. Catalytically, the partially purified glycosyl transferase showed a pH optimum of 9 to 10 and a temperature optimum of 37 degree C. The substrate specificities were also analyzed. UDP-glucose was the best sugar donor with a K(mM) of 0.04 mM which ADP-glucose was not and UDP-galactose was only half as good as UDP-glucose. Among the sugar acceptors tested, acetone cyanohydrin was the best and better than butanone cyanohydrin. Other aliphatic alcohols were poorer sugar acceptors in the following order: isopropanol, butanol, ethanol and glycerol. Phenol was unable to serve as the sugar acceptor for this enzyme. The enzyme was unable to catalyze the transfer of glucose from UDP-glucose to starch, linamarin or simple sugars i.e. glucose, galactose and sucrose. However, a high glucosyltransferase activity was detected in the presence of UDP-glucose and fructose with a K(um) for fructose of 4.4 kM. This activity could be due either to a contaminating UDP-glucose: D-fructose-glucosyltransferase or to cassava glucosyltransferase having two activities, one for linamarin synthesis and the other for sucrose synthesis.ix, 88 leaves : ill.application/pdfengCassavaManihotGlucosyltransferasesMahidol University