Purification And Characterization Of The Trypsin From Euphausia Superba

Antarctic krill (Euphausia superba) is the key species of antarctic ecological system due to its abundance and ecological relevance. Antarctic krill is a shrimp-like crustacean notable for its rapid autolysis post-mortem due to the presence of high concentrations of proteolytic enzymes.The efficient digestion system is based on a combination of enzymes controlled by inhibitors. The efficiency of these enzymes is clearly visualized when the krill post mortemis autolyzed, due to the disabling of the inhibitor system. As a result, the purification of a protease from antarctic krill has significant theoretic and practical importance.In this paper, a trypsin from antarctic krill has been purified, with the procedure of three steps, ammonium sulfate precipitation, DEAE ion-exchange chromatography and affinity chromatography. Sodium dodecyl sulphate polyacrylamide gel electrophoresis shows a20kDa single band of the purified trypsin subunit molecular weight. The Michaelis-Menten constant (Km) and catalytic constant (kcat) of the purified trypsin for the hydrolysis of BApNA were0.108mmol/L and2.62s-1, respectively. The protease has a maximum activity at pH7.0and it is stable in a pH range from7.0to9.0, and shows a high hydrolytic activity at40℃and a high sensitivity at a temperature over55℃. Moreover, the trypsin maintained a lot of activity after incubated for5h at30℃, but lost its activity very quickly when incubated at40℃. At the same time the protease enzyme is stable in alkali environment. Mg2+, Ca2+and K+have a little activation function on the antarctic krill trypsin activity. While Al3+, Cu2+and Zn2+have a strong inhibitory effect. Benzamidine is the protease specific inhibitor. Histidine residue and tryptophan residue are the necessary groups of the enzyme. Arginine residues, amino and sulfydryl may be associated with enzymatic activity, but not the required groups.