DNA sequence analysis, expression, distribution, and physiological role of the Xaa-prolyldipeptidyl aminopeptidase gene fromLactobacillus helveticus CNRZ32

Lactobacillus helveticus CNRZ32 possesses and Xaa-prolyldipeptidyl aminopeptidase (PepX), which releases amino-terminal dipeptides from peptides containing proline residues in the penultimate position. The PepX gene, designatedpepX, fromLb. helveticus CNRZ32 was sequenced. Analysis of the sequence identified a putative 2379-bppepX open-reading frame, which encodes a polypeptide of 793 amino acid residues with a deduced molecular mass of 88 111 Da, The gene shows significant sequence identity with sequencedpepX genes from lactic acid bacteria. The product of the gene contains a motif that is almost identical with the active-site motif of the serine-dependent PepX from lactococci. The introduction ofpepX intoLactococcus lactis LM0230 on either pGK12 (a low-copy-number plasmid vector) did not result in a significant increase in PepX activity, while the introduction ofpepX into CNRZ32 on pGK12 resulted in a four-fold increase in PepX activity. Southern hybridization experiments revealed that thepepX gene from CNRZ32 is well conserved in lactobacilli, pediococci and streptococci. The physiological role of PepX during growth in lactobacillus MRS (a rich medium containing protein hydrolysates along with other ingredients) and milk was examined by comparing growth of CNRZ32 and a CNRZ32 PepX-negative derivative in MRS. However, the CRNZ32 PepX-negative derivative grew in milk at a reduced specific growth rate when compared to wild-type CNRZ32. Introduction of the cloned PepX determinant into the CNRZ32 PepX-negative derivative resulted in a construct with a specific growth rate similar to that of wild-type CNRZ32.