Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in edible burdock (Arctium Iappa L.)

Background: We have previously reported on the variation of total fructooligosaccharides (FOS), total inulooligosaccharides (IOS) and inulin in the roots of burdock stored at different temperatures. During storage at 0°C, an increase of FOS as a result of the hydrolysis of inulin was observed. Moreover, we suggested that an increase of IOS would likely be due to the synthesis of the IOS by fructosyltransfer from 1-kestose to accumulated fructose and elongated fructose oligomers which can act as acceptors for fructan:fructan 1-fructosyltransferase (1-FFT). However, enzymes such as inulinase or fructan 1-exohydorolase (1-FEH) involved in inulin degradation in burdock roots are still not known. Here, we report the isolation and functional analysis of a gene encoding burdock 1-FEH. Results: A cDNA, named aleh1, was obtained by the RACE method following PCR with degenerate primers designed based on amino-acid sequences of FEHs from other plants. The aleh1 encoded a polypeptide of 581 amino acids. The relative molecular mass and isoelectric point (pI) of the deduced polypeptide were calculated to be 65,666 and 4.86. A recombinant protein of aleh1 was produced in Pichia pastoris, and was purified by ion exchange chromatography with DEAE-Sepharose CL-6B, hydrophobic chromatography with Toyopearl HW55S and gel filtration chromatography with Toyopearl HW55S. Purified recombinant protein showed hydrolyzing activity against β-2, 1 type fructans such as 1-kestose, nystose, fructosylnystose and inulin. On the other hand, sucrose, neokestose, 6-kestose and high DP levan were poor substrates. The purified recombinant protein released fructose from sugars extracted from burdock roots. These results indicated that aleh1 encoded 1-FEH