2.8. Lipase assay

Lipase activity was measured spectrophotometrically (410 nm) using p-nitro phenyl dodecanoate (pNPL) as substrate by the method described by Winkler and Stuckmann [22]. In brief, 100 μl of substrate stock solution (0.3% (w/v) pNPL) was added to 1 ml standard reaction buffer (50 mM Na₂HPO₄ pH 7.5, 0.2% (w/v) Na deoxycholate, 0.1% (w/v) gum arabic) and incubated in a water bath with constant shaking at 200 rpm at 22 °C for 5 min. The reaction was initiated by the addition of 2 μl of enzymes and terminated by the addition of 1.2 ml acetone-ethanol (1:1) solution. The reaction duration was 2 min, and the release of p-nitro phenyl (pNP) was recorded at 410 nm using the UV70 spectrophotometer. Enzyme activity was calculated by constructing a standard curve with pNP under the same buffer conditions as the reaction. One unit (U) of lipase activity was defined as the amount of enzyme that liberated 1 μmol of pNP per min under standard assay conditions.

Lipase activity was also measured by the fluorescence-based rhodamine B (RhB) assay using olive oil emulsion [23] with some modifications. An RhB-olive oil emulsion mixture (RhB−OOe) containing 50 mM Na₂HPO₄ (pH 7.5), 1% (w/v) gum arabic, 0.001% (w/v) RhB, and 2% (v/v) olive oil was emulsified with a DrinkMaster for 5 min, and then the pH was adjusted. The enzymatic assays were performed in a 45 mm × 12.5 mm quartz cuvette with magnetic stirring at pH 7.5 and 22 °C using a fluorescence spectrofluorometer (HORIBA Fluorolog, USA). The enzymatic reactions were initiated by the addition of 2 μl of enzyme solution to 1 ml of emulsion. The liberated fatty acids were calculated from the fluorescence emitted at 580 nm (excitation wavelength is 350 nm). The reaction emulsion with heat-denatured enzyme solution was measured in the same way and used as a blank control. A standard curve for oleic acid in the presence of RhB and gum arabic was prepared, and a linear regression was performed allowing the calculation of lipase activity. The fluorescence emission changes were converted into the hydrolysis rate using polynomial equations. One unit (U) of lipase activity was defined as the amount of enzyme releasing 1 μmol of fatty acid per min under the assay conditions.