Lipase assay

Colorimetric assay of lipase using the copper soap method as described by Veerapagu et al. [22]. Fatty acids liberated during hydrolysis of olive oil substrate by lipase can be determined colorimetrically using a cupric acetate-pyridine reagent. The reaction mixture consists of 1 mL of crude enzyme; 2.5 mL of olive oil was incubated for 5 min. Then, the reaction was stopped by adding 1.0 mL of 6 N HCl and 5 mL of benzene. The upper layer 4 mL was pipetted into a test tube and 1.0 mL of cupric acetate-pyridine reagent was added. The pyridine is used to adjust the pH at 6.0–6.2. The free fatty acids (FFA) dissolved in benzene yielding a blue color were determined by measuring the absorbance of benzene solution at 715 nm using spectrophotometer (AZZOTA SV110 Digital Visible Spectrophotometer New Jersey U.S.A). Lipase activity was determined by measuring the amount of FFA from the standard curve of oleic acid.

The amount of lipase that is required to release 1 μmole of fatty acid per minute is defined as one unit of enzyme activity and expressed as units per milliliter.