Evaluation of the potential for use in biocatalysis of a lipase from a wild strain of Bacillus megaterium

• Published in: Journal of molecular catalysis. B, Enzymatic Enzymatic, 2004-10, Vol.31 (1), p.53-61
• Main Authors: Lima, Valéria M.G., Krieger, Nadia, Mitchell, David A., Baratti, Jacques C., Filippis, Ivano de, Fontana, José D.
• Format: Article
• Language: English
• Subjects: Biocatalysis; Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; Lipase; Methods. Procedures. Technologies; Reversed micelles; Stability
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2004.07.005

We evaluated a lipase produced by Bacillus megaterium with respect to its potential for use in biocatalysis in organic solvents. After 72 h of fermentation the enzyme was precipitated directly from the culture broth with the addition of ammonium sulphate to 80% of saturation. It was then resuspended and dialysed. The preparation was shown to hydrolyse triacylglycerides, with further determinations of lipolytic activity being done in aqueous systems through the hydrolysis of p-nitrophenyl palmitate ( pNPP) at 37 °C. The temperature for maximum activity, based on product formation over the first minute of reaction, was 55 °C. The lipolytic activity was reasonably stable during incubation at temperatures of 40–70 °C, with a residual activity of 77% after 10 min incubation at 60 °C. Stability in organic solvents was studied by incubating the enzyme at 29 °C in butanol, toluene, hexane, isooctane and heptane and in various mixtures (25, 50, 80 and 100% (v/v)) of acetone, ethanol and isopropanol in water. Not only was it stable in butanol, hexane and toluene but it actually showed activation after being incubated in increasing percentages of isopropanol, ethanol and acetone, which is quite unusual for lipases. For the hydrolysis of pNPP at 37 °C in AOT (Dioctyl sodium sulfosuccinate)/heptane reversed micelles, the highest specific activities were obtained with W 0 ([H 2O]/[AOT]) values of 5 and 10, being 111 and 104 U mg −1, respectively. The R O/A (ratio of the reaction rates for the same reaction in organic medium and in aqueous medium) was 1.91 for the hydrolysis of pNPP, which is notable because R O/A values for lipases are typically much less than 1.0. The lipase of B. megaterium shows promise for application in biocatalysis due to its good stability at elevated temperatures and in hydrophobic and hydrophilic organic solvents.