Affinity Chromatography of Neutral Metalloendopeptidase Produced by Streptomyces griseoruber on N-Benzyloxycarbonylglycylleucyl-aminohexylamino-Sepharose

A neutral metalloendopeptidase recovered from culture broth of Streptomyces griseoruber was purified by affinity chromatography on N-benzyloxycarbonylglycylleucylaminohexylamino-Sepharose (Z-Gly-Leu-AH-Sepharose) to electrophoretic homogeneity. The enzyme was adsorbed on this adsorbent from phosphate buffer (pH 5.6) and eluted with acetate buffer (pH 4.1) containing 2M urea. The enzyme was inactivated by ethylenediaminetetraacetate but not by sulfhydryl reagents or phenylmethanesulfonyl fluoride. The enzyme showed the maximum caseinolytic activity in the region of pH 6.0-7.0 and was stable within the pH range of 5.0-7.0. The molecular weight was estimated to be 52000. The enzyme preferentially hydrolyzed Z-Gly-Leu-NH2 and Z-Gly-Phe-NH2 among the synthetic substrates tested in this work. Based on taxonomic studies, the producing organism was identified as Streptomyces griseoruber.