Lipase Production and Characterization

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3.), has attracted a lot of attention in recent years because of its diverse biotechnological applications. Lipase occurs widely in nature, but plant lipase, is significant because of its unique substrate selectivity/specificity. Lipase was isolated from the endosperm of 4-day germinated seeds of Cumcumeropsis manii using extraction buffer containing 1.95% (w/v) Tween 80. The crude enzyme was purified through a four step purification procedures of combined ammonium sulphate ((NH 4) SO 4) and cold acetone precipitated proteins, followed by dialysis and repeated gel filtration on sephadex G-200 column chromatography. This yielded enzyme with two activity peaks of lipase indicating the presence of two forms of lipase identified as acidic and alkaline lipases based on their respective pH optimum at 5.9 and 7.5. The acid lipase was found to be stable between pH 4.5 and 6.0 while the alkaline lipase was stable between 6.5 and 8.0 pH ranges. The two lipase fractions showed optimum activities at 37 o C and were found to be stable at 45 o C after incubation for 1 hr. Ca 2+ was observed to be a very good activator and Pb 2+ a potent inhibitor of the two forms of lipases. The acid lipase was purified to 11.0 fold while the alkaline lipase showed 5.4 purification fold. The acid lipase showed a Vmax of 166.7 Unit and Km of 15.67 g L-1 while the alkaline lipase showed Vmax of 142.8 Unit and Km of 13.28 g L-1 .