Trypsin Enzyme from Viscera of Common Kilka (Clupeonella cultriventris caspia): Purification,Characterization, and Its Compatibility with Oxidants and Surfactants |
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Authors: | Abbas Zamani Rasool Madani Mehran Habibi Rezaie |
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Institution: | 1. Department of Fisheries, Faculty of Natural Resources and Environment, Malayer University, Malayer, Hamedan, Iran;2. Department of Biotechnology of Razi Vaccine and Serum Research Institute, Karaj, Alborz, Iran;3. Department of Cell &4. Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran |
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Abstract: | 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-lysine-chloromethylketone (TLCK) inhibitors (p < 0.05). The enzyme was relatively stable toward 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). |
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Keywords: | characterization Clupeonella cultriventris caspia purification trypsin |
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