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JXB Advance Access originally published online on December 5, 2005
Journal of Experimental Botany 2006 57(1):213-223; doi:10.1093/jxb/erj031
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© The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RESEARCH PAPER

Purification, cloning and functional characterization of a fructan 6-exohydrolase from wheat (Triticum aestivum L.)

Liesbet Van Riet1, Vinay Nagaraj2, Wim Van den Ende1, Stefan Clerens3, Andres Wiemken4 and André Van Laere1,*

1Laboratory of Molecular Plant Physiology, Institute of Botany and Microbiology, K.U. Leuven, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium
2The Biodesign Institute at Arizona State University, 9709, ECG 334, Tempe, Arizona 85287-9709, USA
3Laboratory of Neuro-endocrinology and Immunological Biotechnology, Zoological Institute, K.U. Leuven, Naamsestraat 59, B-3000 Leuven, Belgium
4Zurich Basel Plant Science Center, Botanische Institut der Universität Basel, Hebelstrasse 1, CH 4056 Basel, Switzerland

* To whom correspondence should be addressed. E-mail: Andre.Vanlaere{at}bio.kuleuven.ac.be

Fructans, ß2-1 and/or ß2-6 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 ß2-6 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.

Key words: 6-FEH, fructan exohydrolase, fructans, grasses, invertases, levan, Pichia expression, Triticum aestivum, wheat


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