Purification and properties of leucine aminopeptidase from Aspegillus japonica. II.

Abstract

Leucine aminopeptidase was purified from culture filtrate of Aspergillus japonica by calcium acetate treatment, ammonium sulfate fractionation, and column chromatography with diethylaminoethyl (DEAE)-cellulose, Sephadex G-100 and CM-Sephadex C-50. The purified enzyme was homogeneous in disc electrophoretic analysis. Its molecular weight was estimated to be 57000 by Sephadex G-75 gel-filtration. The enzyme was most active at pH 8.0 towards L-leucylglycylglycine (Leu-Gly-Gly) and L-leucyl β-naphthylamide (Leu-β-NA), and its optimum temperature was 50°. The enzyme was stable in a pH range of 5.5 to 8.5 and below 50°. The purified enzyme was highly activated by Co2+ and was strongly inhibited by Fe3+, Hg2+, Cu2+, Pb2+, Ni2+, ethylenediaminetetraaceticacid (EDTA), o-phenanthroline and N-bromosuccinimide. However, it was not affected by SH-reagents and diisopropyl fluorophosphate (DFP). The enzyme is considered to be leucine aminopeptidase, because it preferentially hydrolyzed di-or tripeptides with N-terminal leucine.