Purification and characterization of fructan: fructan fructosyltransferase from Jerusalem artichoke (Helianthus tuberosus L.)


  • Presented at the Second International Conference on Fructan, Aberystwyth, UK, July 1992.


Fructan: fructan fructosyltransferase activity (FFT, EC from Helianthus tuberosus L. was purified 221-fold by a basic procedure involving ammonium sulphate precipitation, lectin chromatography and ion-exchange chromatography. The resulting FFT preparation was separated into three protein bands, of apparent molecular weight 72800, 60 500 and 56 200, by denaturing polyacrylamide gel electrophoresis. These proteins showed affinity to sucrose-Eupergit. FFT proteins with a molecular weight of 72800 were isolated by preparative native gel electrophoresis, and yielded six distinguishable forms on separation by analytical isoelectric focusing. Proteins from the basic purification were separated by preparative isoelectric focusing into several forms with isoelectric points between pH 4.3 and 4.5. Samples from the gel with FFT activity were analyzed by denaturing polyacrylamide gel electrophoresis. Three samples contained only protein with the molecular weight 72800, and one sample contained only protein of apparent molecular weight 60500. The remaining samples contained a mixture of proteins with molecular weights of 72800, 60500 and 56 200.

FFT was detected by 1-kestose-dependent nystose production. The enzyme was most active at pH 65, and up to 80 % of the activity was retained on pre-incubation (1 h) at temperatures of up to 40 °C. FFT transferred fructosyl groups from oligofructans [degree of polymerization (DP) 3–8] of the inulin series. No glycosyl transfer occurred with 6-kestose, neokestose, maltose, raffinose and maltotriose as the sole substrate. Sucrose efficiently accepted fructosyl units from oligofructans with a Km of approximately 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.