Purification and characterization of a novel extracellular carboxylesterase from the moderately halophilic bacterium Thalassobacillus sp. strain DF-E4

An extracellular carboxylesterase from the moderately halophile Thalassobacillus sp. strain DF-E4 was purified to 8-fold with 11% recovery and specific activity of 2,046 U mg−1. The molecular mass of the native enzyme was approximately 49 kDa as determined by analytical ultracentrifugation, while SDS-PAGE analysis showed a single protein band corresponding to a molecular mass of 45 kDa, suggesting that the enzyme was a monomer. Among the pNP (p-nitrophenyl) esters tested, p-nitrophenyl butyrate (C4) was hydrolyzed most effectively, with Km and Vmax values of 0.69 mM and 0.84 μmol min−1 mg−1, respectively, and the optimum activity occurred at pH 8.5, 40°C and 0.5 M NaCl (w/v). The enzyme activity appeared to be stable over pH 6.0–9.5 and up to 45°C for 1 h. Tests of substrate specificity and inhibitor susceptibility revealed it was a serine-type carboxylesterase (EC 3.1.1.1), rather than a lipase. None of the divalent cations tested enhanced the enzyme activity, while most of them had no effect or slightly inhibited the activity. The enzyme activity was strongly inhibited by PMSF, PAO and DEPC, implying that serine, cysteine and histidine residues at the active site were essential for catalysis.