title: Purification, cloning and functional characterization of a fructan 6-exohydrolase from wheat (Triticum aestivum L.).
abstract: Fructans, beta2-1 and/or beta2-6 linked polymers of fructose, are produced by fructosyltransferases (FTs) from sucrose. They are important storage carbohydrates in many plants. Fructan reserves, widely distributed in plants, are believed to be mobilized via fructan exohydrolases (FEHs). The purification, cloning, and functional characterization of a 6-FEH from wheat (Triticum aestivum L.) are reported here. It is the first FEH shown to hydrolyse exclusively beta2-6 bonds found in a fructan-producing plant. The enzyme was purified to homogeneity using ammonium sulphate precipitation, ConA affinity-, ion exchange-, and size exclusion chromatography and yielded a single band of 70 kDa following SDS-PAGE. Sequence information obtained by mass spectrometry of in-gel trypsin digests demonstrated the presence of a single protein. Moreover, these unique peptide sequences, together with some ESTs coding for them, could be used in a RT-PCR based strategy to clone a 1.7 kb cDNA. Functionality tests of the cDNA performed after heterologous expression in the yeast Pichia pastoris showed--as did the native enzyme from wheat--a very high activity of the produced protein against bacterial levan, 6-kestose, and phlein whilst sucrose and inulin were not used as substrates. Therefore the enzyme is a genuine 6-FEH. In contrast to most FEHs from fructan-accumulating plants, this FEH is not inhibited by sucrose. The relative abundance of 6-FEH transcripts in various tissues of wheat was investigated using quantitative RT-PCR.
authors.affiliation: Laboratory of Molecular Plant Physiology, Institute of Botany and Microbiology, K.U. Leuven, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium.
authors.forename: Liesbet
authors.initials: L
authors.lastname: Van Riet
authors.forename: Vinay
authors.initials: V
authors.lastname: Nagaraj
authors.forename: Wim
authors.initials: W
authors.lastname: Van den Ende
authors.forename: Stefan
authors.initials: S
authors.lastname: Clerens
authors.forename: Andres
authors.initials: A
authors.lastname: Wiemken
authors.forename: André
authors.initials: A
authors.lastname: Van Laere
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005630:Fructans / Q000378:metabolism; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D010843:Pichia / Q000235:genetics; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D014908:Triticum / Q000201:enzymology*
chemical_list: D005630:Fructans; D006026:Glycoside Hydrolases