title: Purification and characterization of trypsin from the spleen of tongol tuna (Thunnus tonggol).
abstract: Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin appearing as a single band on native PAGE showed the maximal activity at pH 8.5 and 65 degrees C. It was stable in a wide pH range of 6-11 but unstable at the temperatures greater than 50 degrees C. The enzyme required calcium ion for thermal stability. The activity was strongly inhibited by 1.0 g/L soybean trypsin inhibitor and 5 mM TLCK and partially inhibited by 2 mM ethylenediaminetetraacetic acid. Activity was lowered with an increasing NaCl concentration (0-30%). The enzyme had a Km for Nalpha-p-tosyl-L-arginine methyl ester hydrochloride of 0.25 mM and a Kcat of 200 s-1. The N-terminal amino acid sequence of trypsin was determined as IVGGYECQAHSQPHQVSLNA and was very homologous to other trypsins.
authors.affiliation: Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.
authors.forename: Sappasith
authors.initials: S
authors.lastname: Klomklao
authors.forename: Soottawat
authors.initials: S
authors.lastname: Benjakul
authors.forename: Wonnop
authors.initials: W
authors.lastname: Visessanguan
authors.forename: Hideki
authors.initials: H
authors.lastname: Kishimura
authors.forename: Benjamin K
authors.initials: BK
authors.lastname: Simpson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013154:Spleen / Q000201:enzymology*; D013696:Temperature; D014357:Trypsin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000494:pharmacology; D014413:Tuna
chemical_list: D014361:Trypsin Inhibitors; D014357:Trypsin