An extracellular alpha-galactosidase (Gal27A) with high specific activity of 423 U mg (1) was identified in thermophilic Neosartorya fischeri P1. Its coding gene (1680 bp) was cloned and functionally expressed in Pichia pastoris. Sequence analysis indicated that deduced Gal27A contains a catalytic domain of glycoside hydrolase family 27. The native and recombinant enzymes shared some similar properties, such as pH optima at 4.5, temperature optima at 60-70 degrees C, resistance to most chemicals and saccharides, and great abilities to degrade raffinose and stachyose in soymilk. Considering the high yield (3.1 g L (1)) in P. pastoris, recombinant rGal27A is more favorable for industrial applications. This is the first report on purification and gene cloning of Neosartorya alpha-galactosidase. (C) 2013 Elsevier Ltd. All rights reserved.