title: 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 Ca(2+) (18%) and Mg(2+) (12%) at 30mM. The lipase was inhibited by Co(2+), Cu(2+), Zn(2+), Fe(2) 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 Ca(2+) 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 Ca(2+) alone. The lipase was found to be hydrolytically resistant toward triacylglycerols with more double bonds.
authors.affiliation: Physiology Nutrition and Pathology Division, Central Marine Fisheries Research Institute, Ernakulam North P.O.,P.B. No. 1603, Cochin 682 018, Kerala, India. Electronic address: kajal_iari@yahoo.com.
authors.forename: Kajal
authors.initials: K
authors.lastname: Chakraborty
authors.affiliation: Physiology Nutrition and Pathology Division, Central Marine Fisheries Research Institute, Ernakulam North P.O.,P.B. No. 1603, Cochin 682 018, Kerala, India.
authors.forename: R Paul
authors.initials: RP
authors.lastname: Raj
keywords: Alkaline lipase; Anion exchange chromatography; Bacillus licheniformis MTCC 6824; Sephadex G 100 gel exclusion chromatography