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

• Published in: Acta microbiologica et immunologica Hungarica Vol. 57; no. 3; p. 191
• Main Authors: Verma, M L, Kanwar, S S
• Format: Journal Article
• Language: English
• Published: Hungary 01.09.2010
• Subjects: Bacillus cereus - chemistry; Bacillus cereus - enzymology; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Chromatography, Liquid; Enzyme Stability; Kinetics; Lipase - chemistry; Lipase - isolation & purification; Lipase - metabolism; Molecular Weight; Substrate Specificity
• ISSN: 1217-8950
• DOI: 10.1556/AMicr.57.2010.3.4

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.