| Summary: | 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, cold-adapted Lip4 from Aeromonas sp. revealed well enzymatic activity. In addition, after 60 min incubation between 40-60°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.
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