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Appl Environ Microbiol, May 1998, p. 1831-1836, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Cloning and Characterization of a Prolinase Gene (pepR) from Lactobacillus rhamnosus

Pekka Varmanen,1,* Terhi Rantanen,2 Airi Palva,2 and Soile Tynkkynen1

Research and Development, Valio Ltd., FIN-00370, Helsinki,1 and Agricultural Research Centre of Finland, Food Research Institute, Jokioinen 31600,2 Finland

Received 10 November 1997/Accepted 25 February 1998

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.

* Corresponding author. Mailing address: R&D, Valio Ltd., Meijeritie 4, FIN-00370, Helsinki, Finland. Phone: 358 10381 3126. Fax: 358 10381 3129. E-mail: pekka.varmanen{at}valio.fi.

Appl Environ Microbiol, May 1998, p. 1831-1836, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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