Purification and characterization of a low molecular mass alkaliphilic lipase of Bacillus cereus MTCC 8372

A low molecular mass alkaliphilic extra-cellular lipase of <i>Bacillus cereus</i> MTCC 8372 was purified 35-fold by hydrophobic interaction (Octyl-Sepharose) chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 8 kDa. It is a homopentamer of 40 kDa as revealed by native-PAGE. The lipase was optimally active at 55 °C and retained approximately half of its original activity after 40 min incubation at 55 °C. The enzyme was maximally active at pH 8.5. Mg ²⁺ , Cu ²⁺ , Ca ²⁺ , Hg ²⁺ , Al ³⁺ and Fe ³⁺ at 1 mM enhanced hydrolytic activity of the lipase. Interestingly, Hg ²⁺ ions synergized and Zn ²⁺ and Co ²⁺ ions antagonized the lipase activity. Among surfactants, Tween 80 promoted the lipase activity. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was highly specific towards <i>p</i> -nitrophenyl palmitate and showed a <i>V</i> _max and <i>K</i> _m of 0.70 mmol.mg ⁻¹ .min ⁻¹ and 32 mM for hydrolysis of <i>p</i> NPP.<br /><br />