1Department of Brewing and Fermentation, Junior College of Tokyo University of Agriculture, Tokyo 156-8502, 2Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture, Tokyo 156-8502, Japan
Carboxypeptidases of the intestine of Tilapia nilotica were purified by ammonium sulfate precipitation, followed by trypsin inhibitor-sepharose 4B affinity chromatography, DEAE- and CM-cellulose ion-exchange chromatographies, and polyexchanger PBE 94 chromatofocusing.
Three purified carboxypeptidases were tentatively designated carboxypeptidase I, II and III. Each of the three carboxypeptidases was found to be a single band when examined by poly-acrylamide gel electrophoresis. The purifications of carboxypeptidase I, II and III were 63-, 77- and 79-fold from the crude extract, respectively.
The carboxypeptidases showed the optimum activity at pH8.0 or 8.5 and 30°C, and carboxypeptidase I was specifically found to be able to hydrolyze Bz-Gly-Tyr, while II and III were found to be able to hydrolyze Bz-Gly-Arg. Three enzymes were strongly inhibited by o-phenanthroline. The activities of three enzymes inactivated by o-phenanthroline were regenerated completely by the addition of Co2+, Ni2+ and Zn2+.