Lipase activities were determined by measuring the release of
free fatty acids (FFA) by titration. Standard olive oil emulsion
method [28] was used for olive oil (for activity confirmation
only) and animal fat emulsion method [29] was used for pet
food wastewater. Optimum assay conditions were employed for
hydrolytic experiments with FL and IL. A pH of 7.2 and temperature
of 35 ◦C was used as optimum conditions for FL as
suggested by the supplier (Sigma-Aldrich Pvt. Ltd., MO, USA)
while the pH was adjusted using 0.02M Tris–HCl buffer. The
optimum conditions for IL were found experimentally within the
pH and temperature range of 4–9 and 25–40 ◦C to be at 6.8 and
37 ◦C, respectively. One unit of enzyme activity (U) was defined
as the amount of enzyme, which liberated 1 mol FFA/min
under the assay conditions. Relative activity was defined as the
ratio of activity at any condition to maximum activity of FL at
optimum conditions.