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Purification and Molecular Characterization of a Tripeptidase (PepT) from Lactobacillus helveticus

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Savijoki, Kirsi, Palva, Airi·2000·Applied and Environmental Microbiology
DOI 10.1128/aem.66.2.794-800.2000PMC PMC91898
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Abstract

A tripeptidase (PepT) from a thermophilic dairy starter strain of Lactobacillus helveticus was purified by four chromatographic steps. PepT appeared to be a trimeric metallopeptidase with a molecular mass of 150 kDa. PepT exhibited maximum activity against hydrophobic tripeptides, with the highest activity for Met-Gly-Gly ( K m , 2.6 mM; V max , 80.2 μmol · min −1 · μg −1 ). Some of the hydrophobic dipeptides were slowly hydrolyzed, distinguishing the Lactobacillus PepT from its counterpart in mesophilic Lactococcus lactis . No activity against tetrapeptides or amino acid p -nitroanilide derivatives was observed. The pepT gene and its flanking regions were isolated by PCR and sequenced by cyclic sequencing. The sequence analyses revealed open reading frames (ORFs) 816 bp (ORF1) and 1,239 bp (ORF2) long. ORF2 encoded a 47-kDa PepT protein which exhibited 53% identity with the PepT from L. lactis . The mRNA analyses indicated that pepT conforms a novel operon structure with an ORF1 located upstream. Several putative −35/−10 regions preceded the operon, but only one transcription start site located downstream of the first putative −10 region was identified. An inverted repeat structure with Δ G of −64.8 kJ/mol was found downstream of the PepT-encoding region.

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  • Up until 2003 just 11 peptidases were known to be part of the proteolytic system of L. helveticus: two proline-specific endopeptidases, PepE and PepO; a tripeptidase PepT; four aminopeptidases, PepX, PepI, PepQ, and PepR; and four dipeptidases, PepD, PepV, PepC, and PepN.

    “Up until 2003 just 11 peptidases were known to be part of the proteolytic system of L. helveticus: two proline-specific endopepeptidases, PepE and PepO; a tripeptidase PepT; four aminopeptidases, PepX, PepI, PepQ, and PepR; and four dipeptidases, PepD, PepV, PepC, and PepN (Christensen et al., 1999; Savijoki and Palva, 2000; Kenny et al., 2003; Pan and Tanokur, 2004).”

    From Lactobacillus helveticus: the proteolytic system (2013) · cites this paper as Savijoki and Palva, 2000

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Authors
Savijoki, K., & Palva, A.
Affiliations
  • University of Turku (FI)
  • University of Helsinki (FI)
ORCID
Savijoki, Kirsi
Venue
Applied and Environmental Microbiology
Publisher
American Society for Microbiology
Published
2000-02
Volume
66
Issue
2
Pages
794-800
ISSN
  • 0099-2240 (ISSN-L)
  • 1098-5336
DOI
10.1128/aem.66.2.794-800.2000
PMC
PMC91898
OpenAlex
W2065705785
Cited by
24
References
2
Citations by year
2025: 2 · 2022: 1 · 2019: 2 · 2017: 1 · 2015: 1 · 2013: 2 · 2012: 1
FWCI
0.92
Type
journal article
Language
en
Open access
Licence
journals.asm.org/non-commercial-tdm-license (tdm)
Locations
  • Applied and Environmental Microbiology (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Jukuri (Natural Resources Institute Finland (Luke)) (submittedVersion)
Funders
Academy of Finland
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 › Genetics › Bacterial Genetics and Biotechnology
Keywords
tripeptidase, Lactobacillus helveticus, metalloproteinases, enzyme purification, operon structure, transcription start sites, inverted repeats, Lactococcus lactis, gene sequence analysis
MeSH terms
Amino Acid Sequence, Aminopeptidases, Cloning, Molecular, Lactobacillus, Molecular Sequence Data, Peptide Hydrolases, RNA, Bacterial, RNA, Messenger, Substrate Specificity, Blotting, Northern, Polymerase Chain Reaction, Sequence Analysis, DNA, Reverse Transcriptase Polymerase Chain Reaction

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