Purification of lipase from Geotrichum candidum:: conditions optimized for enzyme production using Box-Behnken design

The fungus Geotrichum candidum was selected from isolates of oil-mill waste as a potent lipase producer. Factors affecting lipase production by the fungus G. candidum in yeast-extract-peptone medium have been optimized by using a Box-Behnken design with seven variables to identify the significant correlation between effects of these variables in the production of the enzyme lipase. The experimental values were found to be in accordance with the predicted values, the correlation coefficient is 0.9957. It was observed that the variables days (6), pH (7.0), temperature (30degreesC), carbon (1.25%), nitrogen (2.0%), Tween (1.0%) and salt concentrations (0.5 mM) were the optimum conditions for maximum lipase production (87.7 LU/ml). The enzyme was purified to homogeneity with an apparent molecular mass of 32 kDa by SDS-PAGE. The optimum pH at 40degreesC was 7.0 and the optimum temperature at pH 7.0 was 40degreesC. The enzyme was stable within a pH range of 6.5 to 8.5 at 30degreesC for 24 h. The enzyme activity was strongly inhibited by AgNO3, NiCl2, HgCl2, and EDTA. However, the presence of Ca2+ and Ba2+ ions enhanced the activity of the enzyme.