Purification and molecular characterization of a tripeptidase (PepT) from Lactobacillus helveticus

A tripeptidase (PepT) from a thermophillic dairy starter strain of Lactobac illus helveticus was purified by four chromatographic steps. PepT appeared to be a trimeric metallopeptidase viith a molecular mass of 150 kDa. PepT e xhibited maximum activity against hydrophobic tripeptides, with the highest activity for Met-Gly-Gly (K-m, 2.6 mM; V-max, 80.2 mu mol . min(-1) . mu g (-1)). Some of the hydrophobic dipeptides were slowly hydrolyzed, distingui shing the Lactobacillus PepT from its counterpart in mesophilic Lactococcus lactis. No activity against tetrapeptides or amino acid p-nitroanilide der ivatives was observed. The pepT gene and its flanking regions were isolated by PCR and sequenced by cyclic sequencing. The sequence analyses revealed open reading frames (ORFs) 816 bp (ORF1) and 1,239 bp (ORF2) long. ORF2 enc oded a 47-kDa PepT protein which exhibited 53% identity with the PepT from L. lactis. The mRNA analyses indicated that pepT conforms a novel operon st ructure with an ORF1 located upstream. Several putative -35/-10 regions pre ceded the operon, but only one transcription start site located downstream of the first putative -10 region was identified. An inverted repeat structu re with Delta G of -64.8 kJ/mol was found downstream of the PepT-encoding r egion.