Aims: The agarase from Thalassomonas agarivorans BCRC 17492 was cloned and overexpressed in Escherichia coli. The characterization of the novel agarase was performed. Methods and Results: The genomic library of T. agarivorans BCRC 17492 was constructed for screening agarase gene. The novel b-agarase, namely AgaB1, was successfully identified and shared only 57% identity to reported agarase from Alteromonas sp. To characterize the AgaB1 protein, the recombinant AgaB1 can be obtained by heterologous expression in E. coli. The agarase activity of AgaB1 was achieved at 30Á25 U per mg at 35°C. According to the analysis of optimal conditions, the highest activity of AgaB1 was attained at 40°C, pH 7Á4 and 200 mmol l À1 NaCl, and half-life of AgaB1 can be maintained for almost 1 h at 40°C. Further determination of substrate hydrolysis indicated that AgaB1 had possession of both endo-and exolytic activity, and neoagarobiose was the major hydrolysis product by TLC and high-performance liquid chromatograph/mass spectrometer (HPLC/MS) analysis. Conclusions:We have successfully cloned and overexpressed the novel b-agarase from T. agarivorans BCRC 17492 in E. coli. The high yield and detailed characterization of recombinant AgaB1 was provided.

Abstract. Thermostable enzymes have the potential as the biocatalyst for industrial applications. To compare the relationship of enzymatic thermostability, the moderately thermophilic and mesophilic bacteria were utilized to explore the properties of esterases. By using the shotgun libraries of mesophilic Thalassomonas agarivorans, and Aeromonas sp., and moderately thermophilic Ralstonia sp., esterases-encoding Lip20, Lip4 and LipRT for α/β-hydrolase fold were cloned, sequenced, and characterized. According to the recombinant proteins overexpressed by Escherichia coli, these results indicated that Lip20, Lip4 and LipRT preferred to hydrolyze short-length p-nitrophenyl (p-NP) esters. The optimal temperature required for the activity of Lip20, Lip4 and LipRT was 30, 40 and 60°C, respectively, corresponding to the trend of bacterial growth temperature. Even at low temperature, coldadapted Lip4 from Aeromonas sp. revealed well enzymatic activity. In addition, after 60 min incubation between 40-60 o C, over 92% residual activity can be retained by the thermostable analysis of LipRT from Ralstonia sp.. Inspecting the predicted structures and amino acid composition, we found that the high helix content was exhibited in LipRT. Also, high frequency residues of Val, Phe and Arg for increasing hydrophobic and salt-bridge interactions were observed. These factors could improve LipRT thermal stabilization and lead to become more rigid.