Neoagaro-oligosaccharides prepared by agar hydrolysis have various application fields, including the pharmaceutical, cosmetic, and food industries. In this study, an agarolytic strain was isolated from a saltwater hot spring and identified as Microbulbifer pacificus LD25 by 16S rRNA. The whole genome sequence of M. pacificus LD25 was obtained. It had a size of 4.27 Mb and comprised 3062 predicted genes in 37 contigs with a G+C content of 58.0%. Six agarases were annotated and classified into three families, namely, GH16 (AgaL1), GH86 (AgaL2, AgaL3), and GH50 (AgaL4, AgaL5, AgaL6), which shared 75-96% identities with unpublished hypothetical proteins and agarases. AgaLl, AgaL4, and AgaL6 can be successfully expressed and purified in Escherichia coli. AgaLl and AgaL4 displayed a significantly agarolytic capability, whereas AgaL6 exhibited a rarely detectable enzymatic activity. The optimal temperature and pH required for the activity of AgaLl and AgaL4 was 50 degrees C and 60 degrees C, respectively, at pH 7. The specific activities of AgaL1 and AgaL(4) were achieved at 16.8 and 9.6 U per mg of protein. Both agarases were significantly inhibited in the presence of EDTA, MgO, ZnCl2, and H2O2. However, AgaLl was resistant to 0.1% SDS and AgaL(4) was slightly activated by CaCl2. Substrate hydrolysis detected by LC-MS/MS analysis indicated that neoagarobiose was the main product during AgaL1 and AgaL4 catalysis. Furthermore, AgaL4 was thermostable and retained over 93% of its relative activity after pre-incubation at 70 degrees C for 180 min. Consequently, M. pacificus LD25 has a potential for agarase production in E. coli and industrial applications. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.