title: 24kDa Trypsin: A predominant protease purified from the viscera of hybrid catfish (Clarias macrocephalus×Clarias gariepinus).
abstract: 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 N(α)-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.
authors.affiliation: Department of Food Science and Technology, Faculty of Technology and Community Development, Thaksin University, Phatthalung Campus, Phatthalung 93110, Thailand. Electronic address: sappasith@tsu.ac.th.
authors.forename: Sappasith
authors.initials: S
authors.lastname: Klomklao
authors.affiliation: Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkla 90112, Thailand.
authors.forename: Soottawat
authors.initials: S
authors.lastname: Benjakul
authors.affiliation: Laboratory of Marine Products and Food Science, Research Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Kishimura
authors.affiliation: Department of Food Technology, School of Agricultural Technology, Walailak University, Nakhon Si Thammarat 80160, Thailand.
authors.forename: Manat
authors.initials: M
authors.lastname: Chaijan
keywords: Isolation; N-terminal amino acid sequence; Purification; Serine proteinase; Trypsin; Viscera