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

Chakraborty, Kajal and Paulraj, R (2010) An extra-cellular alkaline metallolipase from Bacillus licheniformisMTCC 6824: Purification and biochemical characterization. Food Chemistry, 120 (2). pp. 433-442.

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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 41.7 LU/ml of culture medium when the bacterium was cultured for 48 h 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 520 LU/mg after gel exclusion chromatography. The pure enzyme is a monomeric protein and has an apparent molecular mass of 74.8 kDa. The lipase had a Vmax and Km of 0.64 mM/mg/min and 29 mM, 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 82 min at 45 °C, and 48 min at 55 °C. The catalytic activity was enhanced by Ca2+ (18%) and Mg2+ (12%) at 30 mM. The lipase was inhibited by Co2+, Cu2+, Zn2+, Fe2 even at low concentration (10 mM). EDTA, at 70 mM concentration, significantly inhibited the activity of lipase. Phenyl methyl sulfonyl fluoride (PMSF, 70 mM) 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 45 min, as compared to 91.5% when incubated with Ca2+ alone. The lipase was found to be hydrolytically resistant toward triacylglycerols with more double bonds.

Item Type:Article
Official URL/DOI:http://dx.doi.org/10.1016/j.foodchem.2009.10.032
Uncontrolled Keywords:Alkaline lipase; Bacillus licheniformis MTCC 6824; Anion exchange chromatography; Sephadex G 100 gel exclusion chromatography
Subjects:Food Chemistry
Divisions:CMFRI-Cochin > Marine Biotechnology
CMFRI-Cochin > Marine Capture > Crustacean Fisheries
ID Code:4
Deposited By:Madhan M
Deposited On:26 Feb 2010 09:55
Last Modified:26 Oct 2010 15:33

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