Recombinant beta-glycosidases from hyperthermophilic Sulfolobus solfataricus (Ss beta Gly) and Pyrococcus furiosus (CelB) have been characterized with regard to their potential use in lactose hydrolysis at about 70 degrees C or greater. Compared with Ss beta Gly, CelB is approximately 15 times more stable against irreversible denaturation by heat, its operational half-life time at 80 degrees C and pH 5.5 being 22 days. The stability of CelB but not that of Ss beta Gly is decreased 4-fold in the presence of 200 mM lactose at 80 degrees C. CelB displays a broader pH/activity profile than Ss beta Gly, retaining at least 60% enzyme activity between pH 4 and 7. Both enzymes have a similar activation energy for lactose hydrolysis of approximately 75 kJ/mol (pH 5.5), and this is constant between 30 and 95 degrees C. D-Galactose is a weak competitive inhibitor against the release of D-glucose from lactose (K-i approximate to 0.3 M), and at 80 degrees C the ratio of K-i,K-D-galactose to K-m,K-lactose is 2.5 and 4.0 for CelB and Ss beta Gly, respectively. Ss beta Gly is activated up to 2-fold in the presence of D-glucose with respect to the maximum rate of glycosidic bond cleavage, measured with o-nitrophenyl beta-D-galactoside as the substrate. By contrast, CelB is competitively inhibited by D-glucose and has a K-i of 76 mM. The transfer of the galactosyl group from lactose to accepters such as lactose or D-glucose rather than water is significant for both enzymes and depends on the initial lactose concentration as well as the time-dependent substrate/product ratio during batchwise lactose conversion. It is approximately 1.8 times higher for Ss beta Gly, compared with CelB. Overall, CelB and Ss beta Gly share their catalytic properties with much less thermostable beta-glycosidases and thus seem very suitable for lactose hydrolysis at greater than or equal to 70 degrees C.