Purification And Characterization Of Trypsins And Chymotrypsins From Hepatopancreas Of Japanses Sea Bass (Lateolabrax Japonicus)

Trypsin (EC 3.4.21.4) and Chymotrypsin (EC 3.4.21.1) are the large family of serine proteinases, trypsin specifically hydrolyzes peptides at the carboxyl side of arginine and lysine residues, chymotrypsin cleavages the peptide of proteins on the carboxyl side of phenylalanine, tyrosine and tryptophan residues. They play important roles of physiological and biological function in fish, revealing high acitivy at low temperatures and thus have potential applications in food processing. Till now, informations about the purification of fish chymotrypsin are less available, and the study of trypsin focuses on obtaining a deeper insight into their mechanism of physiological and biological functions. In order to study the physiological and biological functions of trypsins and chymotrypsins and its application in food processing, we studied purification, characterization and the physiological and biological functions of the trypsin and chymotrypsin from the hepatopancreas of Japanese sea bass (Lateolabrax japonicus), an important economic fish in China.Trypsins (the cationic form named as trypsin A and the anionic form named as trypsin B) and chymotrypsins (the cationic form named as chymotrypsin A and the anionic form named as chymotrypsin B) from the hepatopancreas of Japanese sea bass (Lateolabrax japonicus) were purified to homogeneity by a series of procedures including ammonium sulfate precipitation and column chromatographies including DEAE-Sepharose, Sephacryl S-200 and Phenyl-Sepharose. SDS-PAGE and Native-PAGE revealed trypsins and chymotrypsins were highly purified. The molecular weights of trypsin A, trypsin B, chymotrypsin A and chymotrypsin B were 21 kDa, 21.5 kDa, 27 kDa and 27.5 kDa, respectively. The characterization study revealed trypsin A and trypsin B both exhibited maximal activity at 40°C and optimal pHs were both 9.0. Chymotrypsin A and chymotrypsin B both exhibited maximal activity at 45°C and optimal pHs were both 8.0. Two-dimensional PAGE (2D-PAGE) analysis exhibited that the isoelectrophoretic points (pI) of chymotrypsin A and B were 8.0 and 7.0. The activity of two trypsins and chymotrypsins were decrease sharply above 50°C and stable up in the pH range from 7.0 to 11.0. The two trypsins and chymotrypsins were effectively inhibited at different degree by serine proteinases inhibitors. The substrate specificity of the two trypsins hydrolysed Boc-Gln-Arg-Arg-MCA most effectively, also, hydrolysis of different substrate were determined in kinetic studies. The kinetic constants showed that trypsin B would be more efficient in catalytic efficiency to most substrates than trypsin A. Using Suc-Leu-Leu-Val-Tyr-MCA as substrate, the Kms of chymotrypsin A and B were 0.8μM and 1.1μM, kcats of the two enzymes were 2.7 s-1 and 2.0 s-1 and the kcat/Km of them were 3.0 (μmol/L) -1 s-1 and 1.9 (μmol/L) -1 s-1, respectively. The N-terminal amino acid sequence of chymotrypsin A and chymotrypsin B were determined as IVNGEEAVPHSWPWQVSLQDY and IVNGEEAVPHSWPWQVSL. Immunoblotting analysis suggested the purified trypsins and chymotrypsins have different degrees of reaction to the anti-crucian carp trypsin and anti-crucian carp chymotrypsin, respectively. Furthermore, the digestive effect of the two trypsins and chymotrypsins on the proteins of shrimp was also investigated.