Purification and characterization of fructan: fructan fructosyl transferase from chicory (Cichorium intybus L.) roots

Abstract

Abstract. Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion-and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 pmol.(mg protein)-l.min-1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization > 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited [3-fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5-50~ Therefore enzyme assays were performed at 0~ The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Key words: Cichorium -Fructan: fructan fructosyl transferase -Fructan -Inulin -1-Kestose Sucrose Abbreviations: Con A = concanavalin A; DP = degree of polymerization; FFT = fructan: fructan fructosyl transferase; Fru = fructose; Glc = glucose; Kes = 1-kestose; MALDI-TOF MS = matrix-assisted laser desorption ionisation time of flight mass spectrometry; Nys = 1,1-nystose; pI = isoelectric point; SST = sucrose: sucrose fructosyl transferase; Sue = sucrose

Transforming a Fructan:Fructan 6G-Fructosyltransferase from Perennial Ryegrass into a Sucrose:Sucrose 1-Fructosyltransferase

“…FTs are very difficult enzymes with respect to kinetic studies, but an estimation of kinetic parameters is sometimes possible (Van den Ende et al, 1996aEnde et al, , 1996bVergauwen et al, 2003) for initial reactions catalyzed by "unifunctional" enzymes and provided that the products formed are not better substrates (compared with the original substrates) or strong inhibitors. The bifunctional Lp6G-FFT/1-FFT enzyme forms a very complex system, since it can catalyze multiple reactions (Fig.…”

“…Therefore, most likely the futile reaction 1K + Suc / Suc + 1K might even be more important than the reaction 1K + Suc / Suc + nK using the same substrates (Lasseur et al, 2006). Overall, it is generally assumed that these futile reactions play a prominent role for all F-type enzymes (Van den Ende et al, 1996b), also explaining why a long lag phase is observed during the in vitro synthesis of longer polymerization fructans by combining S-and F-type enzymes with Suc as a single substrate (Van den Ende and Van Laere, 1996).…”

Fructan 1-Exohydrolases. β-(2,1)-Trimmers during Graminan Biosynthesis in Stems of Wheat? Purification, Characterization, Mass Mapping, and Cloning of Two Fructan 1-Exohydrolase Isoforms,

“…Undissolved material was spun down for 15 min at 40,000g and 4°C. The supernatant was applied to a Con A Sepharose column (25 ϫ 100 mm) and eluted as described (Van den Ende et al, 1996). Active fractions were pooled, adjusted to pH 7.0 with concentrated Tris, and applied on Mono Q1 (HR 5/5, Pharmacia AB, Uppsala) equilibrated with 20 mm Tris-HCl buffer, pH 7.0.…”

“…Active fractions were pooled, adjusted to pH 7.0 with concentrated Tris, and applied on Mono Q1 (HR 5/5, Pharmacia AB, Uppsala) equilibrated with 20 mm Tris-HCl buffer, pH 7.0. Proteins were eluted as described (Van den Ende et al, 1996). 1-FEH w1 (fraction 15) and were diluted five times with 20 mm His-HCl buffer, pH 6.0, and subsequently loaded again on Mono Q2 (1-FEH w1) and Mono Q3 (1-FEH w2), equilibrated with 20 mm His-HCl buffer, pH 6.0.…”

“…Sodium-azide (0.02%, w/v) was added to all buffers to prevent microbial growth. SDS-PAGE was performed on 12.5% (w/v) polyacrylamide gels and stained with Coomassie Brilliant Blue-R250 as described (Van den Ende et al, 1996).…”