Characterization of the exopeptidase activity existing in Theobroma cacao L. during germination

The present work reports exopeptidase activity existing in cacao (Theobroma cacao L.) during germination. Aminopeptidase (APE), carboxypeptidase (CP) and Xaa-Prolyl dipeptidyl aminopeptidase (Xaa-Pro-DAP) membrane-bound enzymes have been identified. The Xaa-Pro-DAP enzyme (E.C. 3.4.14.5) had not been previously detected in germinating cacao seeds. Xaa-Pro-DAP was partially purified and characterized, and the highest activity was found after 10 days of germination. Xaa-Pro-DAP was isolated by precipitation with 40% ammonium sulfate and partially purified with two chromatographic steps. The enzyme had a relative molecular weight of 80kDa as determined by Native-PAGE and was, able to use Ala-Pro-4μβNA as substrate. In the presence of SDS, this enzyme did not show activity because it must be in a trimer to be functional. Its activity was inhibited 44% by the chelating agent EDTA and 48% by the serine peptidase inhibitor PMSF at 0.1mM, indicating that the partially purified Xaa-Pro-DAP is a serine metallopeptidase. The cations Cu²⁺ and Cd²⁺ caused 44% and 67% inhibition, respectively, while the other divalent cations tested had no significant effect on the activity of the partially purified enzyme. The enzyme showed a high specificity for Ala-Pro-pNA as a proteolytic substrate.