The purification of trypsin from the common kilka (Clupeonella cultriventris caspia) viscera (pyloric caeca) resulted in a 28.3-fold increase and 12% yield by ammonium sulfate precipitation (30-60%), Sephadex G-75, and DEAE–cellulose chromatography. Trypsin showed a molecular weight of 23.2 kDa and appeared as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), native-PAGE, and zymography. The trypsin had optimal activity at pH 8.0 and 60 °C for the hydrolysis of α-N-benzoyl-DL-arginine-ρ-nitroanilide hydrochloride (BAPNA) substrate. Trypsin was stable up to 50 °C and at pH range of 7.0-10.0. Activity was significantly inhibited by soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-L-lysinechloromethylketone (TLCK) inhibitors (P < 0.05). The enzyme was relatively stable towards oxidizing agents, retaining 59.7 and 98.0% of its initial activity after 1 h incubation in the presence of 15% H2O2 and 1% sodium perborate, respectively. Trypsin was significantly activated by surfactants and Ca2+, Mg2+, and Mn2+ and inactivated by Fe2+, Zn2+, Cu2+, Al3+, Ba2+, and Co2+ (P < 0.05). Nevertheless, Na+ and K+ had no significant effect on trypsin activity (P > 0.05). The purified trypsin showed significantly higher catalytic efficiency (kcat/Km) than porcine pancreatic trypsin against BAPNA and N-α-p-Tosyl-L-arginine methyl ester hydrochloride (TAME) substrates (P < 0.05).
