Affinity Chromatography of Alkinonase A on N-Carbobenzoxy-Glycyl-Leucyl-Aminohexyl-Sepharose

抄録

N-Carbobenzoxy-glycyl-leucyl-aminohexyl-Sepharose was found to be an effective affinity adsorbent for alkinonase A, an alkaline metalloendopeptidase of Streptomyces violaceorectus. The enzyme was adsorbed on this affinity adsorbent at pH 9.0 and eluted at pH 7.0. The purified enzyme was shown to be homogeneous by polyacrylamide gel electrophoresis, and the molecular weight of the enzyme was estimated to be 35000. The kinetics of the enzyme was studied using N-carbobenzoxy-glycyl-leucine amide as a substrate. The K_m value decreased with increase of pH in the range of 6.0-9.0. The optimum pH for casein hydrolysis was 9.0-9.5, but the specificity rate constant (k_cat / K_m) in Tris-HCl (pH 7.0) was 9 times higher than that in Tris-HCl (pH 9.0) due to the much higher ratio of k_cat values. No remarkable change in the relative rate constant was observed, when the leucine residue was replaced by phenylalanine in N-carbobenzoxy-glycyl-leucine amide. The replacement of the glycine moiety of the peptide with tryptophan or proline, however, markedly decreased the relative rate constant.