PURIFICATION AND CHARACTERIZATION OF FRUCTAN - FRUCTAN FRUCTOSYLTRANSFERASE FROM JERUSALEM-ARTICHOKE (HELIANTHUS-TUBEROSUS L)

Fructan:fructan fructosyltransferase activity (FFT, EC 2.4. 1.100) fro m Helianthus tuberosus L. was purified 221-fold by a basic procedure i nvolving ammonium sulphate precipitation, lectin chromatography and io n-exchange chromatography. The resulting FFT preparation was separated into three protein bands, of apparent molecular weight 72 800, 60 500 and 56 200, by denaturing polyacrylamide gel electrophoresis. These p roteins showed affinity to sucrose-Eupergit. FFT proteins with a molec ular weight of 72800 were isolated by preparative native gel electroph oresis, and yielded six distinguishable forms on separation by analyti cal isoelectric focusing. Proteins from the basic purification were se parated by preparative isoelectric focusing into several forms with is oelectric points between pH 4.3 and 4.5. Samples from the gel with FFT activity were analyzed by denaturing polyacrylamide gel electrophores is. Three samples contained only protein with the molecular weight 728 00, and one sample contained only protein of apparent molecular weight 60500. The remaining samples contained a mixture of proteins with mol ecular weights of 72 800, 60 500 and 5 6 200. FFT was detected by 1-ke stose-dependent nystose production. The enzyme was most active at pH 6 .5, and up to 80% of the activity was retained on pre-incubation (1 h) at temperatures of up to 40-degrees-C. FFT transferred fructosyl grou ps from oligofructans [degree of polymerization (DP) 3-8] of the inuli n series. No glycosyl transfer occurred with 6-kestose, neokestose, ma ltose, raffinose and maltotriose as the sole substrate. Sucrose effici ently accepted fructosyl units from oligofructans with a K(m) of appro ximately 0.2 mM. The rate of fructosyl transfer increased with degree of polymerization (from DP 4). 1-kestose was shown to be an efficient donor of fructosyl units to sucrose.