caa a22 4500 477101720 CHVBK 20180405111543.0 cr unu---uuuuu 170330e19960801xx s 000 0 eng 10.1007/BF00195178 doi (NATIONALLICENCE)springer-10.1007/BF00195178 Purification and characterization of fructan: fructan fructosyl transferase from chicory ( Cichorium intybus L.) roots [Elektronische Daten] [Wim Van den Ende, Dominik Van Wonterghem, Peter Verhaert, Erna Dewil, André Van Laere] 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 μmol·(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 β-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. Springer-Verlag, 1996 Cichorium nationallicence Fructan: fructan fructosyl transferase nationallicence Fructan nationallicence Inulin nationallicence 1-Kestose nationallicence Sucrose nationallicence Con A : concanavalin A nationallicence DP : degree of polymerization nationallicence FFT : fructan: fructan fructosyl transferase nationallicence Fru : fructose nationallicence Glc : glucose nationallicence Kes : 1-kestose nationallicence MALDI-TOF MS : matrix-assisted laser desorption ionisation time of flight mass spectrometry nationallicence Nys : 1,1-nystose nationallicence pI : isoelectric point nationallicence SST : sucrose: sucrose fructosyl transferase nationallicence Suc : sucrose nationallicence Van den Ende Wim Laboratory for Developmental Biology, Botany Institute, Kardinaal Mercierlaan 92, B-3001, Heverlee, Belgium aut Van Wonterghem Dominik Laboratory for Developmental Biology, Botany Institute, Kardinaal Mercierlaan 92, B-3001, Heverlee, Belgium aut Verhaert Peter Laboratory for Developmental Physiology and Molecular Biology, Zoological Institute, Naamsestraat 59, B-3000, Leuven, Belgium aut Dewil Erna Department of Animal Sciences, Physiology of Domestic Animals, Kardinaal Mercierlaan 92, B-3001, Heverlee, Belgium aut Van Laere André Laboratory for Developmental Biology, Botany Institute, Kardinaal Mercierlaan 92, B-3001, Heverlee, Belgium aut Planta Springer-Verlag 199/4(1996-08-01), 493-502 0032-0935 199:4<493 1996 199 425 https://doi.org/10.1007/BF00195178 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00195178 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Van den Ende Wim Laboratory for Developmental Biology, Botany Institute, Kardinaal Mercierlaan 92, B-3001, Heverlee, Belgium aut NATIONALLICENCE 700 1- Van Wonterghem Dominik Laboratory for Developmental Biology, Botany Institute, Kardinaal Mercierlaan 92, B-3001, Heverlee, Belgium aut NATIONALLICENCE 700 1- Verhaert Peter Laboratory for Developmental Physiology and Molecular Biology, Zoological Institute, Naamsestraat 59, B-3000, Leuven, Belgium aut NATIONALLICENCE 700 1- Dewil Erna Department of Animal Sciences, Physiology of Domestic Animals, Kardinaal Mercierlaan 92, B-3001, Heverlee, Belgium aut NATIONALLICENCE 700 1- Van Laere André Laboratory for Developmental Biology, Botany Institute, Kardinaal Mercierlaan 92, B-3001, Heverlee, Belgium aut NATIONALLICENCE 773 0- Planta Springer-Verlag 199/4(1996-08-01), 493-502 0032-0935 199:4<493 1996 199 425 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer