Affinity Chromatography of <italic>Streptomyces erythreus</italic> Trypsin-Like Enzyme on Japanese Quail Ovomucoid

Abstract

For simple, rapid purification of Streptomyces erythreus trypsin-like enzyme (TLE), we examined affinity chromatography with quail ovomucoid (QO) as a ligand of an affinity matrix. We prepared two affinity matrices by the cyanogen bromide method and the oxirane coupling method, and compared their binding efficiencies for TLE using frontal affinity chromatography. The affinity matrix in which QO was immobilized by the cyanogen bromide method showed 30% binding efficiency. However, another matrix, in which QO was immobilized by the oxirane coupling method with the lysine residue of the reactive site blocked by citraconylation, showed 92% binding efficiency. The results suggest that some biologically inactive QO was formed during the coupling reaction by the cyanogen bromide method. When we purified TLE from S. erythreus culture broth by affinity chromatography on QO-Sepharose, TLE was purified about 1,100-fold by affinity chromatography, and was further purified by DEAE-Sephadex A-25 column chromatography to homogeneity. The overall yield of TLE activity was higher than 90%. Thus, we were able to greatly improve previous purification procedures.