Production and partial characterization of a novel thermostable esterase from a thermophilic Bacillus sp.

A thermophilic bacterium, Bacillus sp. 4, newly isolated from Alangüllü thermal spring (Aydın, Turkey), showed a cell-associated esterase activity. Culture conditions in the growth and esterase production by the Bacillus sp. 4 were investigated using partially modified Thermus medium at different pHs (pH 5.00–9.00) and temperatures (50–70 °C). The optimal growth and esterase production was obtained at pH 6.00 and 65 °C. The maximal esterase production was obtained in the mid-stationary phase, and its activity was either intracellular or membrane associated. Optimum pH and temperature for esterase activity were 6.00 and 65 °C, respectively. After 1 and 10 h incubation at 65 °C, the enzyme exhibited approximately 70 and 50% of its original activity, respectively. After 100 h incubation at 40 °C, the original activity of the enzyme was almost protected (83%). The esterase activities were about 99, 100, 100 and 81% of their original values after 1 h incubation at pH 4.00, 6.00, 8.00 and 10.00, respectively. When the pNPB (C4) was used as substrate, the Michaelis–Menten constant (Km) and maximum velocity for the reaction (Vmax) of esterase were 62.89 μM and 833.33 U/mg protein, respectively. Phenylmethanesulphonyl fluoride (PMSF), a serine-specific inhibitor, strongly inhibited the esterase activity, whereas β-mercaptoethanol, a thiol group inhibitor, did not show any effect on the activity. The molecular mass (Mr) of the esterase was estimated to be 81.9 kDa using SDS-PAGE. These results strongly suggest the presence of a single enzyme responsible for pNPB activity in the crude enzyme extract. Of all substrates (C2–C16) tested, the highest activity was towards pNPB, whereas no activity was observed on pNPP (C16).