Anionic trypsin from the spleen of albacore tuna (Thunnus alalunga): Purification, biochemical properties and its application for proteolytic degradation of fish muscle.

Anionic trypsin from albacore tuna spleen was purified by chromatographic separations on Q-Sepharose, Superdex 75 and Arginine Sepharose 4B. The trypsin migrated as single bands in both SDS-PAGE and native-PAGE. The molecular weight of purified trypsin was estimated to be 30 kDa using SDS-PAGE. The enzyme exhibited maximal activity at pH 9.0 and 55 °C for hydrolysis of Boc-Val-Pro-Arg-MCA. pH and temperature stabilities of the trypsin were well maintained in the pH range of 6-11 and over a temperature range from 20 up to 50 °C. The enzyme was effectively inhibited by soybean trypsin inhibitor, N­tosyl­l­phenyl­alanine chloromethyl ketone (TLCK) and Pefabloc SC. The N-terminal amino acid sequence of 20 residues of the purified enzyme was IVGGYECQAHSQPHQVSLNA, which is highly homologous to other fish trypsins. The kcat/Km of the enzyme for Boc-Val-Pro-Arg-MCA was 2.60 ±â€¯0.07 s-1 mM-1. Purified trypsin also hydrolysed fish muscle proteins, suggesting its effectiveness in degradation of food proteins.

Biochemical characterization of trypsins from the hepatopancreas of Japanese sea bass (Lateolabrax japonicus).

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.