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Purification and properties of a neutral invertase from the roots of Cichorium intybus
Author(s) -
Ende Wim,
Laere André
Publication year - 1995
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1995.tb02223.x
Subject(s) - invertase , chemistry , cichorium , fructose , chromatography , sucrose , chromatofocusing , raffinose , cellobiose , size exclusion chromatography , inulin , maltose , hydrolysis , sorbitol , enzyme assay , biochemistry , enzyme , tris , cellulase , biology , agronomy
Multiple activity peaks of neutral invertase (EC 3.2.1.26) 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 μmol (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 HgCl 2 and AgNO 3 and partially inhibited by CoCl 2 , and ZnSO 4 (1 mM). Pyridoxal phosphate (K i ≅ 500 μ M ), Tris (K i ≅ 1.2 m M ), glucose and fructose (K i ≅ 16 m M ) 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.