The genomic DNA and cDNA encoding alpha-L-arabinofuranosidase were cloned from the dimorphic fungus Aureobasidium pullulans ATCC 20524 and sequenced. The open reading frame (2097 bp) of the alpha-L-arabinofuranosidase gene abfB was interrupted by five introns of 49, 49, 50, 65, and 49 bp. The gene encoded a presumed signal peptide of 17 residues and a mature protein of 682 residues with a calculated M-r of 74,230 Da and a theoretical isoelectric point of 4.95. Glu-362 and Glu-440 residues are likely involved in catalytic reactions as an acid/base and a nucleophile, respectively. The protein possessed 15 potential N-glycosylation sites. The deduced amino acid sequence of the abfB gene product was 58% identical to the Penicillium purpurogenum ABF 2, which belongs to the glycoside hydrolase family-51 alpha-L-arabinofuranosidase. The abfB cDNA was functionally expressed in the yeast Pichia pastoris. The recombinant enzyme, AbfB, was purified from the culture filtrate, and it appeared as a single band on SDS-PAGE with an apparent Mr of 110 kDa. AbfB showed alpha-L-arabinofuranosidase activity of 56.6 U/mg of protein toward p-nitrophenyl (pNP) alpha-L-arabinofuranoside at optimal pH 4.5 and 75 degrees C. The enzyme exhibited apparent K-m and V-max, values of 6.27 mM and 78.1 mu mol/mg/min, respectively, for pNP alpha-L-arabinofuranoside. The enzyme was highly active on rye arabinoxylan as well as pNP alpha-L-arabinofuranoside, but it showed weak activity toward alpha-(1 -> 5)-L-arabinobiose, alpha-(1 -> 5)-L-arabinotriose, branched L-arabinan, linear alpha-(1 -> 5)-L-arabinan, and arabinogalactan. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.