Biochemical and molecular characterization of PepR, a dipeptidase, from Lactobacillus helveticus CNRZ32
Open accessAbstract
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.
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The dipeptidase with proline activity (PepR) has been found not to be a limiting enzyme for growth of CNRZ32.
“Similarly, the dipeptidase with proline activity (PepR) has been found not to be a limiting enzyme for growth of CNRZ32 (Shao et al., 1997).”
From Lactobacillus helveticus: the proteolytic system (2013) · cites this paper as Shao et al., 1997
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- Authors
- Shao, W., Yüksel, G. U., Dudley, E. G., Parkin, K. L., & Steele, J. L.
- Affiliations
- University of Wisconsin–Madison (US)
- Department of Food Science, University of Wisconsin-Madison 53706, USA.
- Venue
- Applied and Environmental Microbiology
- Publisher
- American Society for Microbiology
- Published
- 1997-09
- Volume
- 63
- Issue
- 9
- Pages
- 3438-3443
- ISSN
- 0099-2240 (ISSN-L)
- 1098-5336
- PMC
- PMC168651
- OpenAlex
- W1508419681
- Cited by
- 26
- References
- 35
- Citations by year
- 2025: 1 · 2024: 1 · 2020: 1 · 2018: 1 · 2017: 1 · 2015: 1 · 2014: 1 · 2013: 1
- FWCI
- 1.13
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Applied and Environmental Microbiology (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
- Life Sciences › Agricultural and Biological Sciences › Food Science › Probiotics and Fermented Foods
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Hydrolysis and Bioactive Peptides
- Keywords
- dipeptidase, Lactobacillus helveticus, enzyme purification, site-directed mutagenesis, gene replacement, milk acidification, zinc inhibition, enzyme kinetics, amino acid sequence
- MeSH terms
- Amino Acid Sequence, Binding Sites, Dipeptidases, Dipeptides, Enzyme Inhibitors, Hydrogen-Ion Concentration, Isoelectric Point, Lactobacillus, Molecular Sequence Data, Molecular Weight, Substrate Specificity, Zinc, Mutagenesis, Site-Directed
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