Purification and Characterisation of an Aminotripeptidase from Cytoplasm of Lactococcus lactis subsp, cremoris AM2

An aminopeptidase was purified 120-fold with a 78% recovery from the cytoplasm of Lactococcus lactis subsp, cremoris AM2. The purified enzyme exhibited no activity on dipeptides, dipeptideamides, tripeptideamides or tetrapeptides. As activity was observed only with tripeptides, from which it released the N-terminal amino acid, the enzyme was adjudged to be a strict aminotripeptidase. The enzyme had a Mr of 105 000 and showed one band, corresponding to an Mr of 55 000 on SDS-PAGE electrophoresis. Inhibition by EDTA. 8-hydroxyquinoline and 1,10 phenanthroline indicated that the peptidase was a metallo enzyme. Dithiothreitol, bestatin and amastatin caused total inhibition, whereas p-chloromercuribenzoate, bacitracin and phenyl methyl sulphonyl fluoride were without effect. K-m values for tripeptides were within the range 0.18 (Leu-Leu-Leu) to 0.38 mn (Trp-Gly-Gly). Substrate inhibition was noted with some tripeptides at concentrations above 1.5 mM.