2.5. HPL Activity Assay

An aliquot of 0.5 mL of 10 mM linoleic acid solution (pH 6.5) mixed with 0.01 mL of 0.01 M of hydrogen peroxide solution was incubated at 25 °C for 10 min to prepare the substrate fatty acid hydroperoxide. Then, 0.5 mL of AbHPL extract was added, and the reaction system was made up to 3 mL using 0.1 M PBS buffer. The reaction was continued at 25 °C for 30 min. Subsequently, 20 μL of glacial acetic acid, 1 mL of deionized water, and freshly prepared saturated KI solution (0.4 mL) and 1% starch solution (0.1 mL) were added and mixed thoroughly. Soon afterward, the absorbance at 470 nm was measured for 8 consecutive min. One enzyme activity unit was defined as the amount of enzyme that caused a decrease in absorbance of 0.001 per min.