title: Biochemical characterization of trypsins from the hepatopancreas of Japanese sea bass (Lateolabrax japonicus).
abstract: A cationic trypsin (trypsin A) and an anionic trypsin (trypsin B) were highly purified from the hepatopancreas of the Japanese sea bass (Lateolabrax japonicus) by ammonium sulfate precipitation, column chromatographies of DEAE-Sepharose and Sephacryl S-200 HR. Purified trypsins revealed single band on SDS-PAGE and their molecular masses were 21 kDa and 21.5 kDa, respectively. Trypsins A and B exhibited maximal activity at 40°C, and shared the same optimal pH at 9.0 using Boc-Phe-Ser-Arg-MCA as the substrate. The two trypsins were stable up to 45°C and in the pH range from 7.0 to 11.0. Trypsin inhibitors such as Pefabloc SC, PMSF and benzamidine are effective to these two enzymes and their susceptibilities were similar. Apparent K(m)s of trypsins A and B were 1.12 and 0.7 μM and k(cat)s of them were 72.08 and 67.79 S(-1) for Boc-Phe-Ser-Arg-MCA, respectively. The N-terminal amino acid sequences of the two trypsins were determined to the 24th residues, which were highly identical to trypsins from other species of fish while trypsins A and B only shared 45.8% identity. The digestive effect of the two trypsins on native shrimp muscular proteins indicated their effectiveness in the degradation of food proteins.
authors.affiliation: College of Biological Engineering, The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, Xiamen, China.
authors.forename: Qiu-Feng
authors.initials: QF
authors.lastname: Cai
authors.forename: Yu-Kun
authors.initials: YK
authors.lastname: Jiang
authors.forename: Li-Gen
authors.initials: LG
authors.lastname: Zhou
authors.forename: Le-Chang
authors.initials: LC
authors.lastname: Sun
authors.forename: Guang-Ming
authors.initials: GM
authors.lastname: Liu
authors.forename: Kyoshi
authors.initials: K
authors.lastname: Osatomi
authors.forename: Min-Jie
authors.initials: MJ
authors.lastname: Cao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001492:Bass / Q000378:metabolism*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D043143:Hepatopancreas / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000494:pharmacology
chemical_list: D014361:Trypsin Inhibitors; D014357:Trypsin