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24kDa Trypsin: A predominant protease purified from the viscera of hybrid catfish (Clarias macrocephalus×Clarias gariepinus)

Klomklao, Sappasith, Benjakul, Soottawat, Kishimura, Hideki, Chaijan, Manat
Food chemistry 2011 v.129 no.3 pp. 739-746
Clarias gariepinus, Clarias macrocephalus, EDTA (chelating agent), amino acid sequences, ammonium sulfate, animal organs, calcium, catfish, esters, fractionation, heat stability, heat treatment, hybrids, hydrolysis, ions, ketones, molecular weight, pH, polyacrylamide gel electrophoresis, size exclusion chromatography, sodium chloride, soybeans, temperature, trypsin, trypsin inhibitors
Trypsin was purified to homogeneity from the viscera of hybrid catfish (Clarias macrocephalus×Clarias gariepinus) through ammonium sulphate fractionation and a series of chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. It was purified to 47.6-fold with a yield of 12.7%. Based on native-PAGE, the purified trypsin showed a single band. The molecular weight of purified trypsin was estimated as 24kDa by size exclusion chromatography and SDS–PAGE. The optimum pH and temperature for Nᵅ-p-tosyl-l-arginine methyl ester hydrochloride (TAME) hydrolysis were 8.0 and 60°C, respectively. Trypsin was stable to heat treatment up to 50°C, and over a pH range of 6.0–11.0. Trypsin was stabilized by calcium ion. The trypsin activity was strongly inhibited by soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone and partially inhibited by ethylenediaminetetraacetic acid. Activity decreased continuously as NaCl concentration (0–30%) increased. Apparent Kₘ value of trypsin was 0.3mM and Kcₐₜ value was 92.1S⁻¹ for TAME. The N-terminal amino acid sequence of 20 residues of trypsin was IVGGYECQAHSQPPTVSLNA, which is highly homologous with trypsins from other species of fish.