Gewinnung und Charakterisierung von Prolidase aus Escherichia coli B, II. Charakterisierung, Reinigung und Trägerfixierung des Enzyms

Abstract

Prolidase, a specific exopeptidase, is isolated from Escherichia coli B. The enzyme being present in the raw extract is purified and enriched by fractionated ammonium sulfate precipitation, ion exchange chromatography on DEAE‐Sephadex A 50 as well as by gel filtration on Sepharose 4 B. Total yield of prolidase amounts to 19% with a 67fold enrichmentSubstrate specifity of the enzyme mainly corresponds to that of the animal prolidase. It is able to hydrolize the imido linkage at the N‐terminal end of prolin in the case of di‐ and tripeptides. The temperature optimum of prolidase from E. coli B is 37 °C, the pH‐optimum from pH 7.6 to 9.0. Storage stability at pH 8.6 and a temperature of 4 °C is optimal. The enzyme is only active in presence of Mn2+‐ions. This metal cannot be replaced by Mg2‐‐ or Zn2+‐ionsA high enzyme activity and storage stability in presence of Mn2+‐ions can be reached by immobilization of the prolidase, by covalent binding on Sepharose 6 B, adsorption on DEAE‐Sephadex as well as by combination with glutar dialdehyde on DEAE‐Sephadex.