Proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397: purification and characterization.
Proline iminopeptidase (PepIP) is a major peptidase in Lactobacillus delbrueckii subsp. bularicus CNRZ397, encoded by the pepIP gene. Amplification and expression of this gene in Escherichia coli K12 resulted in a very high level of enzyme production. Moreover, export into the E. coli periplasm of 45% of PepIP activity allowed us to purify the enzyme easily by a single ion-exchange chromatography step. PepIP is a trimer of Mr 100000 , composed of three identical subunits. In the presence of 0.1% BSA, PepIP activity was optimal at pH 6-7 and stable at temperatures below 40 degrees C. The enzyme was strongly inhibited by 3,4-dichloroisocoumarin, a serine protease inhibitor, by bestatin and by heavy metal ions. It was also inactivated by p-chloromercuribenzoate, but was reactivated by adding dithiothreitol. PepIP is characterized by a high specificity towards di- or tripeptides with proline at the NH2-terminal position, but is not able to hydrolyse longer peptides, or peptides with hydroxyproline at the NH2-end. The NH2-terminal amino acid sequence of the purified PepIP corresponds to the amino acid sequence deduced from the nucleotide sequence of the pepIP gene.
Gilbert
C
C
Centre de Génétique Moléculaire et Cellulaire, Université Claude Bernard-Lyon, France.
Atlan
D
D
Blanc
B
B
Portalier
R
R
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D007202:Indicators and Reagents; D007477:Ions; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D013379:Substrate Specificity
D007202:Indicators and Reagents; D007477:Ions; D009842:Oligopeptides; D000626:Aminopeptidases; C022450:prolyl aminopeptidase