Agar, a major cell wall component in marine red macroalgae, has recently gained high interest as a potential renewable biomass. By the activity of -agarase which cleaves the -1,4-glycosidic bond, agarose can be hydrolyzed into neoagarobioses which have great potential in the cosmetics, food, and medical industries. Here, based on sequence homology analysis, we isolated a novel exotype -agarase (EXB3) from Gilvimarinus chinensis which have putative glycoside hydrolase (GH) 50 domain. The optimum pH and temperature for the activity of EXB3 were pH 7.0 and 30 degrees C, respectively. The K-m and V-max for agarose were 26 mg/mL and 126.8 U/mg, respectively, and the Kcat/Km value was 3.7 x 10(5) s(-1)M(-1). Under the optimal condition (30 degrees C and pH 7), it was clearly confirmed that neoagarobiose (NA2) was produced as a major product directly from agarose. For the large-scale production of EXB3, we also developed a secretory production platform in Corynebacterium glutamicum. During the fed-batch cultivation in 2 L-scale bioreactor, EXB3 was successfully produced in the culture medium as high as 458.3 mg/L, and EXB3 was purified from the culture supernatant with high purity and recovery yield (24%).