Unveiling aminopeptidase P from Streptomyces lavendulae: molecular cloning, expression and biochemical characterization.

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 ≈60 kDa in Escherichia coli BL21 (DE3). Nucleotide sequencing revealed a 1467 bp 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. K(m) and V(max) of the enzyme were found to be 0.4697 mmol l⁻¹ and 0.6396 μmol min⁻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.