Purification and characterization of fructan: fructan fructosyl transferase from chicory (Cichorium intybus L.) roots

Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 mol·(mg protein) ^–1·min^–1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited -fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Abbreviations Con A concanavalin A - DP degree of polymerization - FFT fructan: fructan fructosyl transferase - Fru fructose - Glc glucose - Kes 1-kestose - MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry - Nys 1,1-nystose - pI isoelectric point - SST sucrose: sucrose fructosyl transferase - Suc sucrose The authors would like to thank E. Nackaerts for valuable assistance. W. Van den Ende is also grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants. P. Verhaert is a research associate of the NFSR. This work was also supported by grant OT/91/18 from the Research Fund K.U. Leuven.