Purification and properties of an intracellular leucine aminopeptidase from the fungus, Penicillium citrinum strain IFO 6352 (vol 17, pg 30, 1996)

An intracellular leucine aminopeptidase (LAP) from Penicillium citrinum (IFO 6352) was purified to homogeneity using three successive purification steps. The enzyme has a native molecular mass of 63 kDa using HPLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity al pH 8.5. An optimum temperature was 45-50 degrees C when L-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60 degrees C. The Michaelis-Menten constants for L-Leu-pNA and L-MET-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl human growth hormone, it showed high specificity for N-terminal methionine residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co2+ ions. The activity of EDTA-treated enzyme was not restored by addition of Zn2+, but reconstitution with Ca2-, Mg2+ or Mn2- restored some enzyme activity. It is likely that Co2+ ions play an important role in the catalysis or stability of the Penicillium citrinum aminopeptidase, as zinc plays a similar function in other leucine aminopeptidases.