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 °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.
Verma
M L
ML
Himachal Pradesh University, Department of Biotechnology, Shimla 171 005, India.
Kanwar
S S
SS
D001409:Bacillus cereus / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002853:Chromatography, Liquid; D004795:Enzyme Stability; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D013379:Substrate Specificity
D001426:Bacterial Proteins; D008049:Lipase