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

A thermophilic bacterium, Bacillus sp. 4, newly isolated from Alangullu thermal spring (Aydin, 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 degrees). The optimal growth and esterase production was obtained at pH 6.00 and 65 degrees 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 degrees C, respectively. After I and 10 h incubation at 65 degrees C, the enzyme exhibited approximately 70 and 50% of its original activity, respectively. After 100 h incubation at 40 degrees 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 I It incubation at pH 4.00, 6.00, 8.00 and 10.00, respectively. When the pNPB (C-4) was used as substrate, the Michaelis-Menten constant (K and maximum velocity for the reaction (V-max) of esterase were 62.89 mu M and 833.33 Wing protein, respectively. Phenylmethanesulphonyl fluoride (PMSF), a serine-specific inhibitor, strongly inhibited the esterase activity, whereas P-mercaptoethanol, a thiol group inhibitor, did not show any effect on the activity. The molecular mass (M-r) 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 (C-2-C-16) tested, the highest activity was towards pNPB, whereas no activity was observed on pNPP (C-16). (c) 2005 Elsevier Inc. All rights reserved.