Characterization of an extracellular thermophilic alkaline esterase produced by Bacillus subtilis DR8806

Abstract

► An alkaline lipolytic metalloenzyme from Bacillus subtilis DR8806 was characterized. ► The optimum pH and temperature of this enzyme were observed to be 8.0 and 50 °C. ► The enzyme showed a preferential specificity for ester of p-nitrophenyl acetate (C 2 ). ► The enzyme exhibited K m of 4.2 mM and V max of 151 μmol min −1 mg −1 to C 2 ester. ► The hydrolysis of fluorescence esters was increased in the presence of the enzyme. This work is a report of the characterization of an alkaline lipolytic enzyme isolated from Bacillus subtilis DR8806. The extracellular extract was concentrated using ammonium sulfate, and ultrafiltration. The active enzyme was purified by Q-sepharose ion exchange chromatography. The molecular mass of the enzyme was estimated to be 60.25 kDa based on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The optimum pH and temperature of this enzyme were observed to be 8.0 and 50 °C, respectively. The enzyme exhibited a half-life of 72 min at its optimum temperature. It was stable in the presence of metal ions (10 mM) such as Ca 2+ , K + and Na + , whereas Cu 2+ , Fe 2+ , Zn 2+ , Mn 2+ , Co 2+ , Mg 2+ and Hg 2+ were found to have inhibitory effects. However, the enzyme activity was not affected significantly by 1% Triton X-100. The study of substrate specificity showed that the purified enzyme has a preferential specificity for small ester of p-nitrophenyl acetate (C 2 ), and it was the most efficiently hydrolyzed substrate as compared to the other esters. The kinetic parameters showed that the enzyme has K m of 4.2 mM and V max of 151 μmol min −1 mg −1 for p-nitrophenyl acetate. The hydrolysis rates of the fluorescence substrates were increased in the presence of the purified enzyme. Regarding the features of the enzyme, it may be utilized as a novel candidate for industrial applications.