Purification and characterization of a lysine aminopeptidase from Kluyveromyces marxianus

Abstract

A lysine aminopeptidase was purified from the yeast Kluyveromyces marxianus. This enzyme was purified 100-fold from a soluble extract obtained at 100,000 g. The purification procedure consisted in fractionated precipitation with ammonium sulfate and five chromatography steps. The native enzyme had a molecular mass of 46 kDa assessed through gel filtration. This aminopeptidase depicted an optimal pH of 7.0 and was stable at a pH range of 4–8, its optimal temperature was 45 °C and the enzyme became unstable at temperatures above 55 °C. The isoelectric point of the purified enzyme was 4.4. Michaelis constant and V max for l-lysine– p-nitroanilide were 0.33 mM and 2.2 mM min −1 per milligram of protein, respectively. The enzyme was strongly inhibited by bestatin, o-phenanthroline and, to a lesser extent, by EDTA, suggesting that this enzyme is a metalloprotease. Our results suggest that the lysine aminopeptidase from Kluyveromyces marxianus might be of biotechnological relevance.