beta-Agarases are mostly categorized into three glycoside hydrolase (GH) families 16, 50, and 86. Recent genomic analysis of Saccharophagus degradans 2-40 revealed the presence of five agarase genes belonging to these GH families. Among the five agarases, Aga50D (a member of GH50) had neither been functionally characterized nor overexpressed. In this report, we present soluble overexpression and molecular characterization of Aga50D. Aga50D was expressed in an active form resulting in a single major product from agarose without intermediates. While known GH50 agarases have both endo-lytic and exo-lytic activities, which produce neoagarobiose as a final product through the intermediate, neoagaro-oligosaccharides, identification and analysis of the reaction product by mass spectrometry and 13C NMR showed that Aga50D had unique exo-lytic activity and was able to produce neoagarobiose directly from agarose. The optimum pH and temperature for the activity were 7.0 and 30 degrees C, respectively. The K (m) and V (max) for agarose were 41.9 mg/ml (4.2 mM) and 17.9 U/mg, respectively.