Lipase production assay using p-Nitrophenyl Palmitate (pNPP) as a substrate

Since lipase claves the p-Nitrophenyl Palmitate (pNPP) substrate, the common pNPP hydrolysis method was used to assess the lipase production. Following Oliveira et al.²⁵ procedure, for each strain, an aliquot (100 µl) of each clear supernatant was added to 900 µl of a reaction mixture with the following composition: 800 µl of 0.25% polyvinyl alcohol solution at pH 6.5 and 100 µl of pNPP solution (3 mM) in isopropanol. The reaction mixtures were incubated for 15 min at 30 °C. After incubation, the reaction was terminated by adding 500 µl of HCl (3 mM) into the mixtures (1:1 v/v). An aliquot (500 µl) of the final two mixtures were added into a 1 ml of NaOH (2 mM). Spectrophotometer (Spectro UV–Vis Double) was used to measure the lipase production at 410 nm. To measure the enzyme activity, a standard curve was used as described previously by Oliveira et al.²⁵. One unit of lipase activity was defined as the amount of lipase required to release 1 µM of pNPP in one minute, under the specified conditions.