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

Verma, M. L. and Kanwar, S. S. (2010) Purification and characterization of a low molecular mass alkaliphilic lipase of Bacillus cereus MTCC 8372. Acta Microbiologica et Immunologica Hungarica, 57 (3). pp. 191-207. ISSN 1217-8950

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Abstract

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 °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. Mg2+, Cu2+, Ca2+, Hg2+, Al3+ and Fe3+ at 1 mM enhanced hydrolytic activity of the lipase. Interestingly, Hg2+ ions synergized and Zn2+ and Co2+ 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 Vmax and Km of 0.70 mmol.mg−1.min−1 and 32 mM for hydrolysis of pNPP.

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