A novel type of arabinoxylan arabinofuranohydrolase isolated from germinated barley - Analysis of substrate preference and specificity by nano-probe NMR

An arabinoxylan arabinofuranohydrolase was isolated from barley malt. The e nzyme preparation, Ara 1, contained two polypeptides with apparent molecula r masses of approximate to 60 and approximate to 66 kDa, a pI of 4.55 and a lmost identical N-terminal amino-acid sequences. With p-nitrophenyl alpha - L-arabinofuranoside (pNPA) as substrate, Ara 1 exhibited a K-m of 0.5 mm an d a V-max of 6.7 mu mol.min(-1).(mg of protein)(-1). Maximum activity was d isplayed at pH 4.2 and 60 degreesC, and, under these conditions, the half-l ife of the enzyme was 8 min. The Ara 1 preparation showed no activity again st p-nitrophenyl alpha -L-arabinopyranoside or p-nitrophenyl beta -D-xylopy ranoside. Substrate preference and specificity were investigated using pure oligosaccharides and analysis by TLC and nano-probe NMR. Ara 1 released ar abinose from high-molecular-mass arabinoxylan and arabinoxylan-derived olig osaccharides but was inactive against linear or branched-chain arabinan. Ar abinose was readily released from both singly and doubly substituted xylo-o ligosaccharides. Whereas single 2-O-linked and 3-O-linked arabinose substit uents on non-reducing terminal xylose were released at similar rates, there was a clear preference for 2-O-linked arabinose on internal xylose residue s. When Ara 1 acted on oligosaccharides with doubly substituted, non-reduci ng terminal xylose, the 3-O-linked arabinose group was preferred as the ini tial point of attack. Oligosaccharides with doubly substituted internal xyl ose were poor substrates and no preference could be determined. The enzyme described here is the first reported arabinoxylan arabinofuranohydrolase wh ich is able to release arabinose from both singly and doubly substituted xy lose, and it hydrolyses p-nitrophenyl alpha -L-arabinofuranoside at a rate similar to that observed for oligosaccharide substrates.