Alkaline proteinase from intestine of Nile tilapia ( Oreochromis niloticus)

An alkaline protease was extracted from the viscera (intestine) of Nile tilapia, Oreochromis niloticus, the second most important fish in Brazilian aquaculture. This enzyme is usually discarded among the tons of waste produced by its processing. The enzyme was purified in three steps: heat treatment, ammonium sulphate fractionation and Sephadex G-75 gel filtration, presenting an yield and purification of 30% and 22-fold, respectively, and showing a single band by SDS-PAGE (23.5 kDa). This enzyme showed Km for the hydrolysis of benzoyl- dl-arginine- p-nitroanilide (BAPNA) equal to 0.755 ± 0.008 mM, an optimum temperature at 50 °C, was stable for 30 min at 50 °C, and optimum pH of 8.0. The protease was strongly inhibited by Al 3+ and Cd 2+, followed by Cu 2+, Hg 2+, Zn 2+ and Co 2+. Inhibition by PMSF and specific trypsin inhibitors provided additional evidences that this activity can be attributed to a trypsin-like enzyme.