Partial purification and characterization of a thermostable trypsin from pyloric caeca of tambaqui (Colossoma macropomum)

A 38.5 kDa alkaline protease from pyloric caeca of tambaqui (Colossoma macr opomum), a tropical freshwater fish, was partially purified in three steps: thermal treatment (45C for 30 min), salting-out (ammonium sulfate at 40-80 % of saturation) and gelfiltration (Sephadex G-75). The purification and yi eld were 51.2-fold and 40%, respectively. The effects of pH, temperature, i nhibitors, and substrates on proteolytic activities of partially purified e nzyme were investigated. The optimum pH was 9.5, while the optimum temperat ure was 60C. This alkaline proteolytic activity remained unaltered after 30 min incubation at 55C. Active site inhibition provided additional evidence that this activity is attributed to a trypsin-like enzyme.