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 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 degrees 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 beta(2-->1) linkages in inulin, Kes and Nys at rates-at least 5 times faster than the beta(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). he enzyme was partially inhibited by Na-EDTA and CaCl2 (1 mM).