PURIFICATION AND CHARACTERIZATION OF A LOW MOLECULAR MASS ALKALIPHILIC LIPASE OF BACILLUS CEREUS MTCC 8372

A low molecular mass alkaliphilic extra-cellular lipase of Bacillus cereus 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 degrees C and retained approximately half of its original activity after 40 min incubation at 55 degrees 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 p-nitrophenyl palmitate and showed a V(max) and K(m) of 0.70 mmol.mg(-1).min(-1) and 32 mM for hydrolysis of pNPP.