Purification, characterisation, cloning and sequencing of the gene encoding oligopeptidase PepO from Streptococcus thermophilus A

Abstract

The oligopeptidase PepO from Streptococcus thermophilus A was purified to protein homogeneity by a five-step chromatography procedure. It was estimated to be a serine metallopeptidase of 70 kDa, with maximal activity at pH 6.5 and 41 degrees C. PepO has endopeptidase activity on oligopeptides composed of between five and 30 amino acids. PepO was demonstrated to be active and stable at the pH, temperature and salt concentrations found in Swiss-type cheese during ripening. Using a battery of PCR techniques, the pepO gene was amplified, subcloned and sequenced, revealing an open reading frame of 1893 nucleotides. The amino acid sequence analysis of the pepO gene-translation product shows homology with PepO enzymes from other lactic acid bacteria and contains the signature sequence of the metallopeptidase family.