Identification and characterisation of the major aspartic proteinase activity in Theobroma cacao seeds

Theobroma cacao seeds contain an unusually high level of aspartic proteinase activity. Although this activity is central to the development of high‐quality cocoa flavour, the T cacao polypeptide responsible has not yet been definitively identified. Here we report the identification and characterisation of an active protein complex from T cacao seeds with an apparent molecular weight of approximately 50 kDa. This active complex contains at least two polypeptides: an approximately 30.5 kDa aspartic proteinase, the product of the TcAP2 gene, and an associated polypeptide, the 20.5 kDa trypsin inhibitor protein. The active complex co‐eluted off a size exclusion column with another complex containing the trypsin inhibitor and a putative acid chitinase. The 30.5 kDa TcAP2 proteinase is apparently a monomeric aspartic proteinase with optimal activity between 42 and 47 °C and an optimal pH of 3.0. Significant inactivation of the TcAP2 activity occurs at acid pH around 47–52 °C, a temperature potentially obtained during cocoa bean fermentation. SDS‐PAGE analysis showed that the purified TcAP2 complex efficiently degrades the cacao seed storage protein vicilin into peptides smaller than 10 kDa. In addition, high‐resolution size exclusion chromatography showed that this proteinase is capable of degrading proteins into peptides as small as di‐ and tripeptides, indicating for the first time that the main T cacao seed aspartic proteinase can produce very small peptide products. Our results demonstrate that the aspartic proteinase encoded by the TcAP2 gene plays a critical role in the production of cocoa flavour precursor peptides during cocoa bean fermentation. Copyright © 2005 Society of Chemical Industry