A 1-FEH II (1-fructan exohydrolase, EC 18.104.22.168) was purified from forced chicory roots (Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A (Con A) affinity chromatography and anion and cation exchange chromatography. This protocol produced a 70-fold purification and a specific activity of 52 nkat mg-1 protein. The apparent size of the enzyme was 60 kDa as estimated by gel filtration and 64 kDa on SDS-PAGE. Optimal activity was found between pH 5.0 and 5.5. The temperature optimum was around 35 °C. No product other than fructose could be detected with inulin as the substrate. The purified enzyme exhibited hyperbolic saturation kinetics with an apparent K(m) of 58 mM for 1-kestose (Kes) and 64 mM for 1,1-nystose (Nys). The purified 1-FEH II hydrolyzed the ̄(2 → 1) linkages in inulin, Kes and Nys at rates at least 5 times faster than the ̄(2 → 6) linkages in levan oligosaccharides and levanbiose. Fructose did not affect the 1-FEH II activity but sucrose (Suc) was a strong inhibitor of this 1-FEH II (K(i) = 5.9 mM). The enzyme was partially inhibited by Na-EDTA and CaCl2 (1 mM).
de Roover, J., van Laere, A., de Winter, M., Timmermans, J. W., & van den Ende, W. (1999). Purification and properties of a second fructan exohydrolase from the roots of Cichorium intybus. Physiologia Plantarum, 106(1), 28-34. https://doi.org/10.1034/j.1399-3054.1999.106104.x