A novel glycoside hydrolase (GH) family 36 alpha-galactosidase gene (designated PtGal36A) from Paecilomyces thermophila was cloned and expressed in Escherichia coli. The deduced sequence of the gene shared the highest identity of 87% with the characterized alpha-galactosidase from Aspergilius nidulans FGSC A4. The recombinant enzyme (PtGal36A) was purified to homogeneity with a purification fold of 11.0 and a recovery yield of 55.2%. PtGal36A was most active at pH 5.0 and 60 degrees C and was stable within the pH range of 4.5-11.5 and up to 50 degrees C. PtGal36A displayed strict specific activity towards substrates with alpha-galactosyl linkages in the nonreducing ends, with the highest activity on stachyose (58.5 U/mg), followed by melibiose (39.2 U/mg) and raffinose (31.4 U/mg). The enzyme efficiently hydrolyzed raffinose family oligosaccharides in soybean meal by more than 95%. Moreover, PtGal36A showed excellent resistance (residual activities > 90%) against alpha-chymotrypsin, proteinase K, subtilisin A, trypsin and papain. Therefore, PtGa136A should be a good candidate for the food and feed industries.