Abstract
Presence of proline residues in the second position of the N-terminus in peptides restricts the usage of many aminopeptidases; however, aminopeptidase P (APP) is capable of removing this blockage. Based on the N-terminal amino acid sequences of APP from Streptomyces lavendulae, app gene was cloned in pET28a(+) and over expressed as a His-tagged protein with a molecular weight of ≈60kDa in Escherichia coli BL21 (DE3). Nucleotide sequencing revealed a 1467bp open reading frame encoding 488 amino acids (NCBI Accession No: GenBank: KC292272.1). The substrate specificity of the recombinant APP was analyzed by the hydrolysis of the Xaa-Pro bond in Gly-Pro dipeptide and bradykinin. Km and Vmax of the enzyme were found to be 0.4697mmoll−1 and 0.6396μmolmin−1, respectively. APP activity was enhanced in the presence of metal ions such as Co2+, Mn2+, Mg2+ and Cu2+ ions and was inhibited by 1,10-phenanthroline, EDTA, PMSF and DTT. The atomic absorption studies revealed the presence of Mn2+ in the protein as a co-factor. This substrate specific metalloenzyme was found to be a tetramer and optimally active at pH 8 and 37°C.
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