CLONING AND CHARACTERIZATION OF A PROLINASE GENE (PEPR) FROM LACTOBACILLUS-RHAMNOSUS

A peptidase gene expressing L-proline-beta-naphthylamide-hydrolyzing a ctivity was cloned from a gene library of lactobacillus rhamnosus 1/6 isolated from cheese. Peptidase-expressing activity was localized in a 1.5-kb SacI fragment. A sequence analysis of the SacI fragment reveal ed the presence of one complete open reading frame (ORF1) that was 903 nucleotides long, The ORF1-encoded 34.2-kDa protein exhibited 68% ide ntity with the PepR protein from Lactobacillus helveticus. Additional sequencing revealed the presence of another open reading frame (ORF2) following pepR; this open reading frame was 459 bp long. Northern (RNA ) and primer extension analyses indicated that pepR is expressed both as a monocistronic transcriptional unit and as a dicistronic transcrip tional unit with ORF2. Gene replacement was used to construct a PepR-n egative strain of L. rhamnosus. PepR was shown to be the primary enzym e capable of hydrolyzing Pro-Leu in L. rhamnosus. However, the PepR-ne gative mutant did not differ from the wild type in its ability to grow and produce acid in milk The cloned pepR expressed activity against d ipeptides with N-terminal proline residues. Also, Met-Ala, Leu-Leu, an d Leu-Gly-Gly and the chromogenic substrates L-leucine-beta-naphthylam ide and L-phenylalanine-beta-naphthylamide were hydrolyzed by the PepR of L. rhamnosus.