An extracellular thermostable alpha-galactosidase from Aspergillus parasiticus MTCC-2796 was purified 16.59-fold by precipitation with acetone, followed by sequential column chromatography with DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found to be a monomeric protein with a molecular weight of about 67.5 kDa. The purified enzyme showed optimum activity against o-nitrophenyl-alpha-D-galactopyranoside (oNPG) at pH 5.0 and a temperature of 50 degrees C. The enzyme was thermostable, showing complete activity even after heating at 65 degrees C for 30 min. The enzyme showed strict substrate specificity for alpha-galactosides and hydrolyzed oNPG (K-m = 0.83 mM), melibiose (K-m = 2.48 mM) and raffinose (K-m = 5.83 mM). Among metal ions and reagents tested, Ca2+ and K+ enhanced the enzymatic activity, but Mg2+, Mn2+, ethylenediaminetetraacetic acid (EDTA) and 2-mercaptoethanol showed no effect, while Ag+, Hg2+ and Co2+ strongly inhibited the activity of the enzyme. The enzyme catalyzed the transglycosylation reaction for the synthesis of melibiose. (C) 2010 Elsevier Ltd. All rights reserved.