title: Cloning and characterization of a prolinase gene (pepR) from Lactobacillus rhamnosus.
abstract: A peptidase gene expressing L-proline-beta-naphthylamide-hydrolyzing activity 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 revealed the presence of one complete open reading frame (ORF1) that was 903 nucleotides long. The ORF1-encoded 34.2-kDa protein exhibited 68% identity 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 transcriptional unit with ORF2. Gene replacement was used to construct a PepR-negative strain of L. rhamnosus. PepR was shown to be the primary enzyme capable of hydrolyzing Pro-Leu in L. rhamnosus. However, the PepR-negative mutant did not differ from the wild type in its ability to grow and produce acid in milk. The cloned pepR expressed activity against dipeptides with N-terminal proline residues. Also, Met-Ala, Leu-Leu, and Leu-Gly-Gly and the chromogenic substrates L-leucine-beta-naphthylamide and L-phenylalanine-beta-naphthylamide were hydrolyzed by the PepR of L. rhamnosus.
authors.affiliation: Research and Development, Valio Ltd., Helsinki, Finland. pekka.varmanen@valio.fi
authors.forename: P
authors.initials: P
authors.lastname: Varmanen
authors.forename: T
authors.initials: T
authors.lastname: Rantanen
authors.forename: A
authors.initials: A
authors.lastname: Palva
authors.forename: S
authors.initials: S
authors.lastname: Tynkkynen
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004150:Dipeptidases / Q000737:chemistry / Q000235:genetics*; D005798:Genes, Bacterial; D007778:Lactobacillus / Q000235:genetics*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D013379:Substrate Specificity
chemical_list: D012333:RNA, Messenger; D004150:Dipeptidases; C022454:prolyl dipeptidase