A psychrotolerant yeast Guehomyces pullulans 17-1 isolated from sea sediment in Antarctica could produce high level (17.2 U/ml) of both extracellular and cell-bound P-galactosidase. The extracellular P-galactosidase in the supernatant of the cell culture of the psychrotolerant yeast G. pullulans 17-1 was purified to homogeneity with a 2.4-fold increase in specific activity as compared to the supernatant by concentration, gel filtration chromatography (Sephadex G-200) and cation-exchange chromatography (CM-Sepharose Fast Flow cation-exchange). The molecular mass of the purified extracellular p-galactosidase was estimated to be 335 kDa. The optimal temperature and pH of the purified beta-galactosidase were 50 C and 4.0, respectively. K-m and V-max values of the purified beta-galactosidase for o-nitrophenyl-beta-D-galactopyranoside were 3.3 mM and 9.2 mu mol/min. Lactose can be converted into glucose and galactose and a large amount of reducing sugar can be released from milk under catalysis of the purified beta-galactosidase. The matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectroscopy identified a peptide ALEEYKK which is the conserved motif of the beta-galactosidases from other yeasts. The results show that the enzyme may have potential applications in food industry. (C) 2010 Elsevier Ltd. All rights reserved.