Affinity Chromatography of Neutral Metalloendopeptidase Produced by Streptomyces mauvecolor on N-Benzyloxycarbonylglycyl-D-leucylaminohexyl-Sepharose

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A neutral metalloendopeptidase, produced by Streptomyces, which has anti-inflammatory activity against carrageenan-induced edema in rats was purified by affinity chromatography on N-benzyloxycarbonylglycyl-D-leucylaminohexyl-Sepharose (Z-Gly-D-Leu-AH-Sepharose) to electrophoretic homogeneity. The enzyme was adsorbed on a Z-Gly-D-Leu-AH-Sepharose column equilibrated with phosphate buffer (pH 7.0). The column was washed with phosphate buffer (pH 5.6) containing 2 M urea and eluted with acetate buffer (pH 4.1) supplemented with 2 M urea. The caseinolytic activity of the enzyme, which was lost on treatment with ethylenediaminetetraacetate, was recovered by the addition of ZnCl_2 to the reaction mixture. The enzyme showed the maximum caseinolytic activity at pH between 7.0 and 8.0,and was stable in the pH range of 5.0-10.0. The molecular weight was estimated to be 59000. The enzyme preferentially hydrolyzed N-benzyloxycarbonylglycyl-L-leucine amide, N-benzyloxycarbonylglycyl-L-phenylalanine amide and N-benzyloxycarbonylglycyl-L-leucyl-L-tyrosine among the synthetic substrates tested in this work. The producing microbe was identified as Streptomyces mauvecolor based on the results of taxonomic studies.
公益社団法人日本薬学会の論文
1985-04-25