A Metalloaminopeptidase from Flavobacterium breve: Characterization and Molecular Cloning

Aminopeptidase from Flavobacterium breve, was purified by a three step FPLC column chromatography to homogeneity from the culture filtrate. The aminopeptidase gene was cloned by using TAIL-PCR technique. The gene encodes for a polypeptide composed of 497 amino acids with a theoretical molecular weight of 58 kDa. SDS-PAGE detection revealed that the protein is of 52 kDa. The native enzyme showed high affinity to Leu-pNA (km 0.0515 mM), and kcat /km of 88.8 s-1mM-1 . The enzyme had an optimum pH 7.5 and was stable from pH 6 to 9. The purified aminopeptidase was stable up to 60 oC and the optimum temperature for the maximum activity was at 70 oC. The amino acid sequence showed 47% identity to aminopeptidase of Aeromonas caviae (family M14), a Zn2+ dependent metallozyme.