The purification and characterization of an extracellular alpha-L-arabinofuranosidase (alpha-L-AFase) from Chaetomium sp. was investigated in this report. The alpha-L-AFase was purified to homogeneity with a purification fold of 1030. The purified alpha-L-AFase had a specific activity of 20.6 U mg(-1). The molecular mass of the enzyme was estimated to be 52.9 kDa and 51.6 kDa by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.0 and 70 degrees C, respectively. The enzyme was stable over a broad pH range of 4.0-10.0 and also exhibited excellent thermostability, i.e., the residual activities reached 75% after treatment at 60 degrees C for 1 h. The enzyme showed strict substrate specificity for the alpha-L-arabinofuranosyl linkage. The K-m and V-max values for p-nitrophenyl (pNP)-alpha-L-arabinofuranoside were calculated to be 1.43 mM and 68.3 mu mol min(-1) mg(-1) protein, respectively. Furthermore, the gene encoding alpha-L-AFase was cloned and sequenced and found to contain a catalytic domain belonging to the glycoside hydrolase (GH) family 43 alpha-L-AFase. The deduced amino acid sequence of the gene showed the highest identity (67%) to the putative alpha-L-AFase from Neurospora crassa. This is the first report on the purification, characterization and gene sequence of an alpha-L-AFase from Chaetomium sp. (C) 2011 Elsevier Ltd. All rights reserved.