• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of aemPermissionsJournals.ASM.orgJournalAEM ArticleJournal InfoAuthorsReviewers
Appl Environ Microbiol. Sep 1997; 63(9): 3438–3443.
PMCID: PMC168651

Biochemical and molecular characterization of PepR, a dipeptidase, from Lactobacillus helveticus CNRZ32.

Abstract

A dipeptidase with prolinase activity from Lactobacillus helveticus CNRZ32, which was designated PepR, was purified to gel electrophoretic homogeneity and characterized. The NH2-terminal amino acid sequence of the purified protein had 96% identity to the deduced NH2-terminal amino acid sequence of the pepR gene, which was previously designated pepPN, from L. helveticus CNRZ32. The purified enzyme hydrolyzed Pro-Met, Thr-Leu, and Ser-Phe as well as dipeptides containing neutral, nonpolar amino acid residues at the amino terminus. Purified PepR was determined to have a molecular mass of 125 kDa with subunits of 33 kDa. The isoelectric point of the enzyme was determined to be 4.5. The optimal reaction conditions, as determined with Pro-Leu as substrate, were pH 6.0 to 6.5 and 45 to 50 degrees C. The purified PepR had a Km of 4.9 to 5.2 mM and a Vmax of 260 to 270 mumol of protein per min/mg at pH 6.5 and 37 degrees C. The activity of purified PepR was inhibited by Zn2+ but not by other cations or cysteine, serine, aspartic, or metal-containing protease inhibitors or reducing agents. Results obtained by site-directed mutagenesis indicated that PepR is a serine-dependent protease. Gene replacement was employed to construct a PepR-deficient derivative of CNRZ32. This mutant did not differ from the wild-type strain in its ability to acidify milk. However, the PepR-deficient construct was determined to have reduced dipeptidase activity compared to the wild-type strain with all dipeptide substrates examined.

Full Text

The Full Text of this article is available as a PDF (283K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Anderson DG, McKay LL. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol. 1983 Sep;46(3):549–552. [PMC free article] [PubMed]
  • Atlan D, Gilbert C, Blanc B, Portalier R. Cloning, sequencing and characterization of the pepIP gene encoding a proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397. Microbiology. 1994 Mar;140(Pt 3):527–535. [PubMed]
  • Bairoch A. The PROSITE dictionary of sites and patterns in proteins, its current status. Nucleic Acids Res. 1993 Jul 1;21(13):3097–3103. [PMC free article] [PubMed]
  • Bhowmik T, Fernández L, Steele JL. Gene replacement in Lactobacillus helveticus. J Bacteriol. 1993 Oct;175(19):6341–6344. [PMC free article] [PubMed]
  • Chen JD, Morrison DA. Cloning of Streptococcus pneumoniae DNA fragments in Escherichia coli requires vectors protected by strong transcriptional terminators. Gene. 1987;55(2-3):179–187. [PubMed]
  • Chich JF, Chapot-Chartier MP, Ribadeau-Dumas B, Gripon JC. Identification of the active site serine of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis. FEBS Lett. 1992 Dec 14;314(2):139–142. [PubMed]
  • Chopin A. Organization and regulation of genes for amino acid biosynthesis in lactic acid bacteria. FEMS Microbiol Rev. 1993 Sep;12(1-3):21–37. [PubMed]
  • Christensen JE, Lin DL, Palva A, Steele JL. Sequence analysis, distribution and expression of an aminopeptidase N-encoding gene from Lactobacillus helveticus CNRZ32. Gene. 1995 Mar 21;155(1):89–93. [PubMed]
  • Dao ML, Ferretti JJ. Streptococcus-Escherichia coli shuttle vector pSA3 and its use in the cloning of streptococcal genes. Appl Environ Microbiol. 1985 Jan;49(1):115–119. [PMC free article] [PubMed]
  • Doi E, Shibata D, Matoba T. Modified colorimetric ninhydrin methods for peptidase assay. Anal Biochem. 1981 Nov 15;118(1):173–184. [PubMed]
  • Dudley EG, Husgen AC, He W, Steele JL. Sequencing, distribution, and inactivation of the dipeptidase A gene (pepDA) from Lactobacillus helveticus CNRZ32. J Bacteriol. 1996 Feb;178(3):701–704. [PMC free article] [PubMed]
  • Dudley EG, Steele JL. Nucleotide sequence and distribution of the pepPN gene from Lactobacillus helveticus CNRZ32. FEMS Microbiol Lett. 1994 Jun 1;119(1-2):41–45. [PubMed]
  • Dunny GM, Lee LN, LeBlanc DJ. Improved electroporation and cloning vector system for gram-positive bacteria. Appl Environ Microbiol. 1991 Apr;57(4):1194–1201. [PMC free article] [PubMed]
  • Englard S, Seifter S. Precipitation techniques. Methods Enzymol. 1990;182:285–300. [PubMed]
  • Fenster KM, Parkin KL, Steele JL. Characterization of a thiol-dependent endopeptidase from Lactobacillus helveticus CNRZ32. J Bacteriol. 1997 Apr;179(8):2529–2533. [PMC free article] [PubMed]
  • Fernández L, Bhowmik T, Steele JL. Characterization of the Lactobacillus helveticus CNRZ32 pepC gene. Appl Environ Microbiol. 1994 Jan;60(1):333–336. [PMC free article] [PubMed]
  • Juillard V, Le Bars D, Kunji ER, Konings WN, Gripon JC, Richard J. Oligopeptides are the main source of nitrogen for Lactococcus lactis during growth in milk. Appl Environ Microbiol. 1995 Aug;61(8):3024–3030. [PMC free article] [PubMed]
  • Khalid NM, Marth EH. Purification and Partial Characterization of a Prolyl-Dipeptidyl Aminopeptidase from Lactobacillus helveticus CNRZ 32. Appl Environ Microbiol. 1990 Feb;56(2):381–388. [PMC free article] [PubMed]
  • Kitazono A, Ito K, Yoshimoto T. Prolyl aminopeptidase is not a sulfhydryl enzyme: identification of the active serine residue by site-directed mutagenesis. J Biochem. 1994 Nov;116(5):943–945. [PubMed]
  • Klein JR, Dick A, Schick J, Matern HT, Henrich B, Plapp R. Molecular cloning and DNA sequence analysis of pepL, a leucyl aminopeptidase gene from Lactobacillus delbrueckii subsp. lactis DSM7290. Eur J Biochem. 1995 Mar 15;228(3):570–578. [PubMed]
  • Klein JR, Schmidt U, Plapp R. Cloning, heterologous expression, and sequencing of a novel proline iminopeptidase gene, pepI, from Lactobacillus delbrueckii subsp. lactis DSM 7290. Microbiology. 1994 May;140(Pt 5):1133–1139. [PubMed]
  • Miller CG, Schwartz G. Peptidase-deficient mutants of Escherichia coli. J Bacteriol. 1978 Aug;135(2):603–611. [PMC free article] [PubMed]
  • Varmanen P, Rantanen T, Palva A. An operon from Lactobacillus helveticus composed of a proline iminopeptidase gene (pepI) and two genes coding for putative members of the ABC transporter family of proteins. Microbiology. 1996 Dec;142(Pt 12):3459–3468. [PubMed]
  • Varmanen P, Steele J, Palva A. Characterization of a prolinase gene and its product and an adjacent ABC transporter gene from Lactobacillus helveticus. Microbiology. 1996 Apr;142(Pt 4):809–816. [PubMed]
  • Walter R, Simmons WH, Yoshimoto T. Proline specific endo- and exopeptidases. Mol Cell Biochem. 1980 Apr 18;30(2):111–127. [PubMed]
  • Yüksel GU, Steele JL. DNA sequence analysis, expression, distribution, and physiological role of the Xaa-prolyldipeptidyl aminopeptidase gene from Lactobacillus helveticus CNRZ32. Appl Microbiol Biotechnol. 1996 Feb;44(6):766–773. [PubMed]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...