Purification and characterization of aminopeptidase (pumAPE) from Ustilago maydis

Abstract

The aminopeptidase pumAPE was purified from the haploid fungus Ustilago maydis FB1 strain. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included anion-exchange, hydrophobic interaction, and gel filtration chromatography, resulting in a 23% recovery. The molecular mass of the dimeric enzyme was estimated to be 110 kDa and 58 kDa by gel filtration chromatography and SDS–PAGE, respectively. Enzymatic activity was optimal at pH 7.0 and at 35 °C toward Lys- pNA and the p I was determined to be 5.1. The enzyme was inhibited by EDTA–Na 2, 1,10- phenanthroline, bestantin, PMSF and several divalent cations (Cu 2+, Hg 2+ and Zn 2+). The aminopeptidase showed a preference for lysine and arginine in the N-position. The K m value was 54.4 μM and the V max value was 408 μmol min −1 mg −1 for Lys-pNA.