Characterization and mass spectrometry analysis of aminopeptidase N from Pseudomonas putida Lup

An intracellular aminopeptidase N synthesized by Pseudomonas putida Lup was purified and characterized. The approx. 150-fold purified enzyme showed highest activity against A-β-naphthylamide at pH 7.5 and at temperature 40°C and was 100% thermostable for 240 min at 40°C. P. putida lup aminopeptidase N is a monomer with molecular mass approx. 99 kDa determined by SDS-PAGE and gel permeation chromatography. The enzyme has broad substrate specificity, but is the most active against protein substrates with N-terminal alanine and arginine. The activity of P. putida Lup aminopeptidase N is strongly inhibited in the presence of specific metallopeptidase inhibitors and is partly recovered in the presence of Zn²⁺ and Co²⁺ ions. Co²⁺, Mg²⁺ and Ca²⁺ ions increased the activity of the enzyme. Moreover, the enzyme was inhibited by inhibitors of cysteine enzymes. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to PepN of Pseudomonas putida GB-1.