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

Abstract

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°C. No product other than fructose could be detected with inulin as the substrate. The purified enzyme exhibited hyperbolic saturation kinetics with an apparent Km of 58 mM for 1‐kestose (Kes) and 64 mM for 1,1‐nystose (Nys). The purified 1‐FEH II hydrolyzed the β(2?1) linkages in inulin, Kes and Nys at rates at least 5 times faster than the β(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 (Ki=5.9 mM). The enzyme was partially inhibited by Na‐EDTA and CaCl2 (1 mM).

Fructan degradation and hydrolytic activity in tuberous roots of Viguiera discolor Baker (Asteraceae), a herbaceous species from the cerrado

“…Affinity chromatography on Sepharose Con-A retained enzyme activity ( figure 6) showing that FEH is a glycoprotein as found for the same enzyme extracted from different plants including species from Poaceae and Asteraceae families (Bonnett et al 1994, De Roover et al 1999b). Elution of the activity was obtained with 40 mM methyl-α-D-mannopyranoside, the maximal activity (peak) obtained being of 0.924 µmoles of fructose.mL .…”

“…These facts suggest that 1-FEH might be activated by the reduction of the photoassimilates supply and/or in periods of increased demand for energy. More recently Van den Ende et al (2001) considered the 1-FEH II purified from tuberous roots of Cichorium intybus L. (De Roover et al 1999b) as a "survival" enzyme due to the fact that it was strongly induced in the roots mainly after defoliation, when energy demands greatly increased.…”

“…Despite the importance of determining the properties of the enzymes involved in fructan depolymerisation for the understanding of fructan breakdown, only a few 1-FEHs from dicots have been purified to electrophoretic homogeneity (Claessens et al1990, Marx et al 1997, De Roover et al 1999b, Henson 2000.…”

Fructan Biosynthetic and Breakdown Enzymes in Dicots Evolved From Different Invertases. Expression of Fructan Genes Throughout Chicory Development

“…Although the activity is well characterized in various plant species [43,44], only a few plant FEH enzymes have been purified to electrophoretic homogeneity. They include three fructan 1-exohydrolases from chicory (1-FEH I, IIa, and IIb) [45,46,47], a 1-FEH from H. tuberosus [48], a fructan 6-exohydrolase (6-FEH) from L. perenne [49], a FEH that preferentially hydrolyzes β(2→6) linkages (Avena sativa [50]), and another FEH that is able to attack both β(2→1) and β(2→6) linkages (Hordeum vulgare[51]). Chicory 1-FEH I differs only slightly from the 1-FEH II iso-enzymes in its kinetic properties and inhibition by sucrose [46].…”

Produção de frutanos em calos e plântulas clonadas in vitro de Viguiera discolor Baker (Asteraceae)

“…Por sua vez, os frutanos de cadeias longas da série da inulina são de difícil obtenção, existindo poucas espécies vegetais capazes de sintetizá-los, paralelamente a um controle restrito da ação de enzimas hidrolíticas durante os processos de armazenamento nos órgãos de reserva (De Roover et al 1999).…”