beta-Galactosidase (lactase), which catalyzes the hydrolysis of lactose into glucose and galactose, is one of the most important enzymes used in dairy processing. In this study, a gene that encoded an extremely thermostable beta-galactosidase from Pyrococcus furiosus (Pflactase) was cloned and expressed in Escherichia coil BL21. The recombinant enzyme was purified by heat treatment and Ni-NTA affinity chromatography. The enzyme displayed optimal activity at 90 degrees C and pH 7.0 in phosphate buffer. The specific activity of the recombinant enzyme on o-nitrophenyl-beta-D-galactopyranoside was 10.2 U/mg at 0 degrees C and 130.0DU/mg at 90 degrees C. The half-lives of the enzyme were 31423.4, 8168.3, 4017.7, 547A, 309.6, and 203.5 min at 70 degrees C, 80 degrees C, 85 degrees C, 90 degrees C, 95 degrees C, and 100 degrees C, respectively. The recombinant enzyme exhibited both beta-galactosidase and beta-glucosidase activity. The active inclusion bodies of beta-galactosidase were easily isolated by nonionic detergent treatment and directly used for lactose conversion in a repetitive batch mode. More than 54% (90 degrees C) or 88% (10 degrees C) of the original enzyme activity was retained after 10 conversion cycles under optimum conditions. These results suggest that the recombinant thermostable beta-galactosidase may be suitable for the hydrolysis of lactose in milk processing. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.