PROLINE IMINOPEPTIDASE FROM LACTOBACILLUS-DELBRUECKII SUBSP BULGARICUS CNRZ-397 - PURIFICATION AND CHARACTERIZATION

Proline iminopeptidase (PepIP) is a major peptidase in Lactobacillus d elbrueckii subsp. bulgaricus CNRZ397, encoded by the pepIP gene. Ampli fication 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 enz yme easily by a single ion-exchange chromatography step. PepIP is a tr imer of M(r) 100 000, composed of three identical subunits. In the pre sence 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 in activated by p-chloromercuribenzoa te, but was reactivated by adding dithiothreitol. PepIP is characteriz ed by a high specificity towards di- or tripeptides with proline at th e NH2-terminal position, but is not able to hydrolyse longer peptides, or peptides with hydroxyproline at the NH2-end. The NH2-terminal amin o acid sequence of the purified PepIP corresponds to the amino acid se quence deduced from the nucleotide sequence of the pepIP gene.