[45] Extracellular Aminopeptidase from Clostridium histolyticum

Clostridial aminopeptidase (CAP) is an extracellular enzyme of broad substrate specificity that has been isolated from the culture filtrate of Clostridium histolyticum and purified to homogeneity. This chapter describes the procedure followed based on the assay principle: The enzyme cleaves efficiently various N-terminal amino acid residues, including proline, from peptides of low and of high molecular weight. The tripeptide Pro-Gly-Pro, which is resistant to most proteolytic enzymes, is used as a selective substrate for the routine assay during the purification of CAP. Proline and Gly-Pro are produced as the only products and the amount of proline formed is determined colorimetrically by the acid ninhydrin method. It describes the purification procedure starting from Cultivation of Bacteria and Preparation of the Culture Filtrate. It also explains the key properties of the aminopeptidase, including its physical and kinetic properties and its stability.