Physiologia plantarum vol:93 issue:2 pages:241-248
Multiple activity peaks of neutral invertase (EC 18.104.22.168) were found in chicory roots (Cichorium intybus L. var. foliosum cv. Flash). The main activity peak was purified by a combination of anion-exchange chromatography, hydrophobic interaction chromatography, chromatofocusing and gel filtration. This protocol produced a 77-fold purification and a specific activity of 1.6 mu mol (mg protein)(-1) min(-1). The mass of the enzyme was 260 kDa as estimated by gel filtration and 65 kDa on SDS-PAGE. Optimal activity was found between pH 7 and 7.5. The purified enzyme exhibited hyperbolic saturation kinetics with a K-m between 10 and 20 mM for sucrose. No other products than glucose and fructose could be detected. Raffinose was hydrolyzed at a rate of 2.4% relative to sucrose whereas the enzyme did not hydrolyze maltose, cellobiose, trehalose, 1-kestose, 1,1-nystose or inulin. Neutral invertase activity was completely inhibited by HgCl2 and AgNO3 and partially inhibited by CoCl2 and ZnSO4 (1 mM). Pyridoxal phosphate (K-i approximate to 500 mu M), Tris (K-i approximate to 1.2 mM), glucose and fructose (K-i approximate to 16 mM) were strong inhibitors of the enzyme. Fructose and Tris behaved as competitive inhibitors. A possible role for the enzyme's activity in vivo is discussed.