An extra-cellular alkaline metallolipase from Bacillus licheniformisMTCC 6824: Purification and biochemical characterization

Abstract

An extra-cellular lipase produced by Bacillus licheniformis MTCC 6824 was purified to homogeneity by ammonium sulphate fractionation, ethanol/ether precipitation, dialysis, followed by anion-exchange chromatography on Amberlite IRA 410 (Cl− form) and gel exclusion chromatography on Sephadex G 100 using Tris–HCl buffer (pH 8.0). The crude lipase extract had an activity of 41.7LU/ml of culture medium when the bacterium was cultured for 48h at 37°C and pH 8.0 with nutrient broth supplemented with sardine oil as carbon source. The enzyme was purified 208-fold with 8.36% recovery and a specific activity of 520LU/mg after gel exclusion chromatography. The pure enzyme is a monomeric protein and has an apparent molecular mass of 74.8kDa. The lipase had a Vmax and Km of 0.64mM/mg/min and 29mM, respectively, with 4-nitro phenylpalmitate as a substrate, as calculated from the Lineweaver–Burk plot. The lipase exhibited optimum activity at 45°C and pH 8.0, respectively. The enzyme had half-lives (T1/2) of 82min at 45°C, and 48min at 55°C. The catalytic activity was enhanced by Ca2+ (18%) and Mg2+ (12%) at 30mM. The lipase was inhibited by Co2+, Cu2+, Zn2+, Fe2 even at low concentration (10mM). EDTA, at 70mM concentration, significantly inhibited the activity of lipase. Phenyl methyl sulfonyl fluoride (PMSF, 70mM) completely inactivated the original lipase. A combination of Ca2+ and sorbitol induced a synergistic effect on the activity of lipase with a significantly high residual activity (100%), even after 45min, as compared to 91.5% when incubated with Ca2+ alone. The lipase was found to be hydrolytically resistant toward triacylglycerols with more double bonds.