Partial purification and biochemical characterization of an extremely thermo- and pH-stable esterase with great substrate affinity

Abstract

An esterase from a thermophilic bacterium, Geobacillus sp. DF20, was partially purified. Final purification factor was found to be 64.5-fold using Q-Sepharose ion exchange column chromatography. Native polyacrylamide gel electrophoresis indicated the presence of a single active esterase. The substrate specificity of this esterase was high for p-nitrophenyl butyrate (pNPB) substrate. The optimum pH and temperature for the enzyme activity were 7.0 and 50 ^°C, respectively. The pH and heat stability profiles show that this enzyme is more stable under neutral conditions at 50 ^°C. K_m and V_{max} values for this esterase acting on pNPB were 0.12 mM and 54.6 U/mg protein, respectively. Presence of 10 % (v/v) acetonitrile in the reaction medium indicated that purified enzyme was strongly inhibited. It was also detected that some metal ions affected enzyme activity at different rates. As a result, it was observed that esterase from Geobacillus sp. DF20 has extreme temperature and pH stabilities. Therefore, the stability and K_m value of the enzyme make this study interesting when compared with the literature.