beta-1,6-glucan is a polysaccharide found in brown macroalgae and fungal cell walls. In this study, a beta-1,6-endoglucanase gene from Saccharophagus degradans 2-40(T), gly30B, was cloned and overexpressed in Escherichia coli. Gly30B, which belongs to the glycoside hydrolase family 30 (GH30), was found to possess beta-1,6-endoglucanase activity by hydrolyzing beta-1,6-glycosidic linkages of pustulan (beta-1,6-glucan derived from fungal cell walls) and laminarin (beta-1,3-glucan with beta-1,6-branchings, derived from brown macroalgae) to produce gentiobiose and glucose as the final products. The optimal pH and temperature for Gly30B activity were found to be pH 7.0 and 40 A degrees C, respectively. The kinetic constants of Gly30B, V (max), K (M), and k (cat) were determined to be 153.8 U/mg protein, 24.2 g/L, and 135.6 s(-1) for pustulan and 32.8 U/mg protein, 100.8 g/L, and 28.9 s(-1) for laminarin, respectively. To our knowledge, Gly30B is the first beta-1,6-endoglucanase characterized from bacteria. Gly30B can be used to hydrolyze beta-1,6-glucans of brown algae or fungal cell walls for producing gentiobiose as a high-value sugar and glucose as a fermentable sugar.