The second cc-galactosidase gene (designated as RmgalB) was cloned from the thermophilic fungus Rhizomucor miehei and expressed in Pichia pastoris. The gene belonging to glycoside hydrolase (GH) family 36 has an open reading frame (ORF) of 2241 bp encoding 746 amino acids with two introns. The recombinant cc-galactosidase (RmgalB) was secreted at high levels of 1953.9 U ml(-1) in high cell density fermentor, which is the highest yield obtained for a cc-galactosidase. The purified enzyme as a tetramer gave a single band corresponding to a molecular mass of 83.1 kDa in SDS-PAGE. The enzyme exhibited a very high specific activity of 505.5 U mg(-1). The optimum temperature and pH of RmgalB were determined to be 55 degrees C and pH 5.5, respectively. It was stable within pH 5.5-9.5 and up to 55 degrees C. RmgalB displayed specificity toward raffinose and stachyose, and completely hydrolyzed the anti-nutritive raffinose family oligosaccharides (RFOs). These properties make RmgalB useful in the food and feed industries. (C) 2015 Elsevier Inc. All rights reserved.