Purification and characterization of an aminopeptidase (Pep C-like) from Lactobacillus casei subsp. casei IFPL 731 isolated from raw goat's milk cheese

摘要

A Pep C-like aminopeptidase was purified from the cell-free extract of Lactobacillus casei subsp. casei IFPL 731 by ammonium sulphate precipitation, hydrophobic interaction chromatography, anion-exchange chromatography, chromatofocusing and gel filtration. SDS-polyacrylamide gel electrophoresis showed one protein band of 50 kDa. The enzyme was a 200 kDa tetramer under non-reducing conditions. Activity was optimal at pH 7.5 and 55°C and was inhibited by the thiol-group inhibitor, p-hydroxymercuribenzoate. Enzyme activity was strongly inhibited by 0.1 mM Cu2+. The aminopeptidase hydrolyzed a wide range of p-nitroanilide derivatives and several dipeptides and tripeptides, showing a preference for substrates containing hydrophobic residues at the N-terminal position. The enzyme showed a high affinity for Leu-pNA (K(m), 0.05 mM).