Purification and molecular characterization of a tripeptidase (PepT) from Lactobacillus helveticus.
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 micromol. min(-1). microg(-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 DeltaG of -64.8 kJ/mol was found downstream of the PepT-encoding region.
Savijoki
K
K
Agricultural Research Centre of Finland, Food Research Institute, Jokioinen 31600, Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland. kirsi.savijoki@mtt.fi
Palva
A
A
D000595:Amino Acid Sequence; D000626:Aminopeptidases; D015152:Blotting, Northern; D003001:Cloning, Molecular; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D012329:RNA, Bacterial; D012333:RNA, Messenger; D010447:Peptide Hydrolases; D000626:Aminopeptidases; C023191:PepT tripeptidase