Purification and properties of a second fructan exohydrolase from the roots of Cichorium intybus

A 1-FEH II (1-fructan exohydrolase, EC 3.2.1.80) was purified from forced c hicory roots (Cichorium intybus L. var. foliosum cv. Flash) by a Combinatio n of ammonium sulfate precipitation, concanavalin A (Con A) affinity chroma tography and anion and cation exchange chromatography. This protocol produc ed 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 degrees C. No product other than fru ctose could be detected with inulin as the substrate, The purified enzyme e xhibited 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 hydro lyzed the beta(2-->1) linkages in inulin, Kes and Nys at rates-at least 5 t imes faster than the beta(2-->6) linkages in levan oligosaccharides and lev anbiose. Fructose did not affect the 1-FEH II activity but sucrose (Suc) wa s a strong inhibitor of this 1-FEH II (K-i=5.9 mM). he enzyme was partially inhibited by Na-EDTA and CaCl2 (1 mM).