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

Abstract

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, N‑tosyl‑l‑phenyl‑alanine 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 ± 0.07 s-1 mM-1. Purified trypsin also hydrolysed fish muscle proteins, suggesting its effectiveness in degradation of food proteins.