Purification, characterization, gene cloning, sequencing, and overexpression of aminopeptidase N from Streptococcus thermophilus A

The general aminopeptidase PepN from Streptococcus thermophilus A was purif ied to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtr ation chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys-7-amino-4-methylcoumarin at pH 7 and 3 7 degrees C. It was strongly inhibited by metal chelating agents, suggestin g that it is a metallopeptidase, The activity was greatly restored by the b ivalent cations Co2+, Zn2+, and Mn2+, Except for proline, glycine, and acid ic amino acid residues, PepN has a broad specificity on the N-terminal amin o acid of small peptides, but no significant endopeptidase activity has bee n detected. The N-terminal and short internal amino acid sequences of purif ied PepN were determined. By using synthetic primers and a battery of PCR t echniques, the pepN gene was amplified, subcloned, and further sequenced, r evealing an open reading frame of 2,541 nucleotides encoding a protein of 8 47 amino acids with a molecular weight of 96,252, Amino acid sequence analy sis of the pepN gene translation product shows high homology with other Pep N enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter -based expression plasmid and the 452-fold overproduced PepN enzyme was pur ified to homogeneity from the periplasmic extract of the host Escherichia c oli strain. The overproduced enzyme showed the same catalytic characteristi cs as the wild type enzyme.