author(s)
Biswajit Mohanty

abstract
The proteolytic enzyme trypsin was purified from the gastrointestinal tract waste of grass carp Ctenopharyngodon idella (Valenciennes, 1844) with ammonium sulfate fractionation (ASF) followed by DEAE-cellulose chromatography, and Benzamidine Sepharose fast flow column affinity chromatography. Trypsin was purified 26.2-fold with an 11.1% yield. The purified enzyme was active between pH 9.0 and 11.0, and maximal activity of the enzyme was observed at pH 10.0. Highest activity was found at 60°C. The activity was reduced further after reaching the maximum activity point of temperature. The trypsin enzymatic activity was decreased by 40% and 60%, when incubated at 90°C for 30 min. The Km, Kcat, and catalytic efficiency values of purified trypsin were obtained is 0.062 mM and 19.23/s, and 310.16/s/mM. Degree of hydrolysis (DH) of the proteases on muscle protein increased with increase of enzyme concentrations. The enzyme activity was also further inhibited by SBTI, PMSF, and N-α-p-tosyl-L-lysine chloromethyl ketone. The molecular weight of the purified enzyme was obtained to be 20.2 kDa by SDS-PAGE. The study showed that trypsin from grass carp visceral waste of could find use in applications where maximum activity at moderate temperature is desired.