A novel type of arabinoxylan arabinofuranohydrolase isolated from germinated barley

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Abstract
An arabinoxylan arabinofuranohydrolase was isolated from barley malt. The enzyme preparation, Ara 1, contained two polypeptides with apparent molecular masses of ≈ 60 and ≈ 66 kDa, a pI of 4.55 and almost identical N-terminal amino-acid sequences. With p-nitrophenyl α-l-arabinofuranoside (pNPA) as substrate, Ara 1 exhibited a Km of 0.5 mm and a Vmax of 6.7 µmol·min−1·(mg of protein)−1. Maximum activity was displayed at pH 4.2 and 60 °C, and, under these conditions, the half-life of the enzyme was 8 min. The Ara 1 preparation showed no activity against p-nitrophenyl α-l-arabinopyranoside or p-nitrophenyl β-d-xylopyranoside. Substrate preference and specificity were investigated using pure oligosaccharides and analysis by TLC and nano-probe NMR. Ara 1 released arabinose from high-molecular-mass arabinoxylan and arabinoxylan-derived oligosaccharides but was inactive against linear or branched-chain arabinan. Arabinose was readily released from both singly and doubly substituted xylo-oligosaccharides. Whereas single 2-O-linked and 3-O-linked arabinose substituents on non-reducing terminal xylose were released at similar rates, there was a clear preference for 2-O-linked arabinose on internal xylose residues. When Ara 1 acted on oligosaccharides with doubly substituted, non-reducing terminal xylose, the 3-O-linked arabinose group was preferred as the initial point of attack. Oligosaccharides with doubly substituted internal xylose were poor substrates and no preference could be determined. The enzyme described here is the first reported arabinoxylan arabinofuranohydrolase which is able to release arabinose from both singly and doubly substituted xylose, and it hydrolyses p-nitrophenyl α-l-arabinofuranoside at a rate similar to that observed for oligosaccharide substrates.