The gene encoding a thermostable beta-galactosidase from Sulfolobus solfataricus was cloned and expressed in Escherichia coli. We investigated the enzymatic production of lactulose from lactose and fructose by this thermostable beta-galactosidase. The optimum pH and temperature for lactulose kproduction by the beta-galactosidase were 6.0 and 80 degrees C, respectively. In thermostability experiments, the enzyme followed first-order kinetics of thermal inactivation, and the half-lives of the enzyme at 75, 80, 85, 90, and 95 degrees C were 91, 48, 35, 2.6, and 0.72 h, respectively, suggesting that the enzyme is highly stable up to 85 degrees C. Enzyme activity was increased by the addition of Fe2+ and decreased by Ba2+. The optimum substrate and enzyme concentrations were 40% (w/v) lactose and 20% (w/v) fructose at 3.0 units/ml, respectively. Under the optimum conditions, the beta-galactosidase from S. solfataricus produced approximately 50 g/l lactulose in 6 h of reaction time with a productivity of 8.3 g/l h. (c) 2006 Elsevier Inc. All rights reserved.