A putative proline iminopeptidase of Thermotoga maritima is a leucine aminopeptidese with lysine-p-nitroanilide hydrolyzing activity

A putative aminopeptidase P gene (TM0042, Swissport Q9WXP9, GeneBank T. maritima aminopeptidase P enzyme, gave a homogenous protein band with an apparent molecular weight of 40 kDa in SDS-PAGE analysis. The enzyme was purified 23-fold with the specific activity of 16.5 unit/mg with the final recovery of 22%. The enzyme was thermostable up to 90 °C for 30 min. An optimal activity was observed at 90 °C at pH 7.5. The purified enzyme was stable between pH 6.5 and 8 at 80 °C with the optimum of pH 7.5. Based on the amino acid sequence, the enzyme belongs to M 24B family of metalloenzymes. None of the divalent cations enhance the activity of the enzyme while Pb 2+ , Cu 2+ , Co 2+ , Cd 2+ , and Zn 2+ were inhibitory to the enzyme activity. Divalent cation of Mg 2+ showed 100% enzyme activity, to a lesser extent, Ca 2+ and Mn 2+ whereas strong inhibition of enzyme activity was observed with Zn 2+ and Cd 2+ . The enzyme designated as putative aminopeptidase P was very low activity in hydrolyzing proline- p -nitroanilide. Kinetic studies on the purified enzyme confirmed that the enzyme is a leucine aminopeptidase. Enzyme also hydrolyzes lysine- p -nitroanilide with efficiency comparable to that of leucine- p -nitroanilide. This is the first report of leucine aminopeptidase with lysine- p -nitroanilide hydrolyzing activity, which belongs to the M 24B family of metalloenzymes.