Seasonal variation of fructan‐β‐fructosidase (FEH) activity and characterization of a β‐(2‐1)‐linkage specific FEH from tubers of Jerusalem artichoke (Helianthus tuberosus)

Abstract

SUMMARYThe fructan-y?-fructosidase activity (1-FEH; EC 3.2.1.80) that degrades inulin in tubers oi Helianthus tuberosus L. appears to be developmentally regulated; it was low in growing tubers but increased during dormancy and sprouting. In spite of relatively high 1-FEH activity in vitro, fructose concentration was very low in developing and dormant tubers and increased markedly only during sprouting. A fructan-/S-fructosidase from such sprouting tubers was purified 41-fold to a single protein band on one-dimensional sodium dodecylsulphate-polyacrylamide gels. The purification procedure included ammonium sulphate precipitation, lectin-affinity chromatography on concanavalin A, anion-exchange and cation-exchange chromatography. The enzyme had an apparent molecular mass of 75000 measured by size-exclusion chromatography, and 79000 measured by one-dimensional sodium dodecylsulphate-polyacrylamide gel electrophoresis. It exhibited a high substrate specificity, hydrolysing terminal /?-(2-l)-fructosyl-fructose-linkages in linear and branched fructan oligomers; y^-(2-6)-linkages were hardly hydrolysed. Hydrolysis of inulin oligomers followed normal saturation kinetics: K^ values for 1,1-kestotetraose and 1,1,1-kestopentaose were 8-3 mM and 12 mM, respectively. Fructosyl residues were hydrolysed from inulin oligomers by a multi-chain mechanism. The fructan-y?-fructosidase showed optimal enzyme activity at pH 5-2, and it retained its full activity after pre-incubation for 1 h at up to 40 °C. The release of fructose from 5 mM 1,1-kestotetraose was reduced by 25 % when 1-FEH was assayed in the presence of 10 mM sucrose. It is proposed that the inhibition of 1-FEH activity by sucrose is a mechanism for controlling fructan degradation in planta.

Hydrolysis of fructan in grasses: a β‐(2‐6)‐linkage specific fructan‐β‐fructosidase from stubble of Lolium perenne

“…4). Indeed, the linkage specificity of a 1-FEH (Marx et al, 1997) and that of fructosyl transferases (Luscher, Frehner & Nosberger, 1993a, fe;Simmen et al, 1993) were characterized successfully using a similar approach.…”

“…Inulin oligosaccharides (1-kestotriose, 1,1 kestotetraose and 1,1,1-kestopentaose) were prepared from NeosugarP®; 6(}-kestotriose from Allium cepa bulbs; and 6-kestotriose, (l&6)-kestotetraose and 6,6-kestotetraose from T. aestivum stems, as described previously (Marx et al, 1997). Oligomers of DP > 7 were prepared from T. aestivum stem extracts (Marx et al, 1997).…”

“…Inulin oligosaccharides (1-kestotriose, 1,1 kestotetraose and 1,1,1-kestopentaose) were prepared from NeosugarP®; 6(}-kestotriose from Allium cepa bulbs; and 6-kestotriose, (l&6)-kestotetraose and 6,6-kestotetraose from T. aestivum stems, as described previously (Marx et al, 1997). Oligomers of DP > 7 were prepared from T. aestivum stem extracts (Marx et al, 1997). Fructan oligosaccharides that had eluted in the void volume after size-exclusion chromatography (Superperformance column, 1 X 200 cm, Merck, Basel, Switzerland) were concentrated, re-dissolved and desalted, using successive layers of Dowex 1 (H"*"), polyvinyl-polypyrrolidone and Dowex 50 (HCO3") ion-exchanger (Bachmann, Matile & Keller, 1994) and then concentrated to dryness in vacuo.…”

“…Sugars from enzyme assays were separated and quantified by HPAEC-PAD, as described by Marx et al (1997).…”

“…Up to now, however, only FEH from roots of Chicorium intybus (Claessens, Van Laere & De Proft, 1990) and Helianthus tuberosus (Marx, Nosberger & Frehner, 1997) has been purified to apparent homogeneity as revealed by silver-stained gels from lD-SDS-PAGE. In monocots, FEH has been partly purified from the stem bases of Dactylis glomerata (Yamamoto & Mino, 1985) and Hordeum vulgare (Henson, 1989), from the shoots of Lolium temulentum (Simpson, Walker & Pollock, 1991) and several fractions from Lolium rigidum (Bonnett & Simpson, 1993.…”