Optimal immobilization of trypsin from the spleen of albacore tuna (Thunnus alalunga) and its characterization.

Trypsin purified from the spleen of albacore tuna was immobilized onto Octyl Sepharose CL-4B, glutaraldehyde activated silica and 5'-4,4'-dimethyltryptamine-thymidine-succinyl controlled pore glass. Trypsin was highly and efficiently immobilized onto Octyl Sepharose CL-4B, with the highest activity (6.26 U/g support) and specific activity (1.45 U/mg bound protein). The optimum conditions for trypsin immobilization onto Octyl Sepharose CL-4B were 40 mg/mL trypsin solution, pH 7 at 4 °C for 6 h of incubation time. The optimal temperature and pH for the hydrolysis of N-α-benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA) by the immobilized trypsin were 55 °C and 8.5, both of which were higher than that of the free form. In comparison with free enzyme, the immobilized trypsin exhibited greater resistances against thermal inactivation and organic solvents. The immobilized enzyme was less sensitive to inhibition by the soybean trypsin inhibitor compared with the free soluble form of the enzyme. According to the results, the immobilized trypsin and free enzyme retained 83% and 47% of their activity, respectively, when they were incubated with 1 µM of the soybean trypsin inhibitor. For the reusability study, the immobilized trypsin maintained 60% of its activity after 4 periods of activity, indicating that the immobilized trypsin had appropriate stability and could be reused.

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.