Purification And Characterization Of Trypsin And Chymotrypsins From Japanese Eel(Anguilla Japonica)

With the active site as serine, the major digestive proteases of trypsin(EC 3.4.21.4) and chymotrypsin(EC 3.4.21.1) belong to the family of serine proteinases, both of them are endopeptidases. Trypsin specifically cleaves peptide bonds on the carboxyl side of lysine and arginine residues whereas the chymotrypsin specifically cleaves the peptide bonds on the carboxyl side of hydrophobic amino acids, such as phenylalanine, tyrosine, and tryptophan residues(aromatic amino acids). Together with chymotrypsin, trypsin play important role on physiological and digestive function in fish. Significantly, trypsin and chymotrypsin could retain high activity at low-temperature make it possible to be used as tool enzyme for food, pharmacy, biotechnology industries. In the present, trypsins have received more attention than that of chymotrypsins both at home and abroad. The research considered the fresh water Japanese eel(Anguilla japonica) as target and aimed to isolate and purify the trypsin and chymotrypsin simultaneously from the hepatopancreas and study their enzymatic characteristics in detail, and also aimed to provide the theoretically fundamental information for their application.In the present study, trypsin(anionic nature, named for Trypsin B) and chymotrypsin(cationic nature named for chymotrypsin A and anionic nature named for chymotrypsin B) were purified to homogeneity from the hepatopancreas of Japanese eel by ammonium sulfate precipitation and column chromatographies of DEAE-Sepharose, Phenyl-Sepharose, Sephacryl S-200 HR and SP-Sepharose.SDS-PAGE and Native-PAGE revealed that both trysin B and chymotrypsins were monomer form, and were highly purified. Using Boc-Phe-Ser-Arg-MCA as substrate, the trypsin B with a molecular weight of 21.5 k Da revealed optimal activity at 40 °C, p H 8.5. The kinetic experiments showed that Km value of trypsin for hydrolysis of Boc-Phe-Ser-Arg-MCA was 3.1 μmol/L, kcat value of trypsin was 59.9(S-1) and catalytic efficiency(kcat/Km) was 19.3(μmol/L)-1 S-1.The molecular weight of the purified trypsin and chymotrypsin A and chymotrypsin B were 27 and 27.5 k Da, respectively. Using Suc-Leu-Leu-Val-Tyr-MCA as subtrate, the optimal temperature of chymotrypsin A and chymotrypsin B were 40 and 45 °C, the optimal p H were 8 and 8.5, respectively. The kinetic experiments showed that Km values of chymotrypsin A and chymotrypsin B for hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA were 9.4 and 9.1 μmol/L, respectively, kcat values of chymotrypsin A and chymotrypsin B were 1.8 and 4.1(S-1), respectively, and the catalytic efficiency(kcat/Km) of chymotrypsin A and chymotrypsin B were 0.2 and 0.5(μmol/L)-1 S-1, respectively. The enzymes were not stable at acidic condition, however, desmonstrated strong stability at alkaline condition.Their activities were significantly inhibited by Cu2+, Zn2+ and Fe2+. Serine proteinase inhibitors, such as PMSF, benzamidine, and STI, strongly inhibited the activity of the trypsin and chymotrypsins. The N-terminal 12 amino acid sequence of trypsin was determinated as IVGGYECEPHSQ, exhibiting high identity to trypsins from other fish species. Western blot analysis demonstrated that the trypsin and chymotrypsins reacted positively with rabbit anti-crucian carp trypsin and chymotrypsin B polyclonal antibodies. Furthermore, both trypsin B and chymotrypsin B exhibited degradation ability toward shrimp muscular proteins, suggesting their effectiveness in the digestion of food proteins.