Abstract
Microbial alkaline protease inhibitor, S-SI, was immobilized by covalent binding with Sepharose (agarose spheres) which was previously activated by cyanogen bromide. S-SI- Sepharose, thus obtained, contained 7.2mg of S-SI in 1ml of settled volume, and its subtilisin-combining capacity was 16.6mg per ml. Stability of S-SI did not be lowered by immobilization. Affinity of immobilized S-SI for various proteases was examined, and it was revealed that α-chymotrypsin, as well as microbial alkaline proteases, had affinity for immobilized S-SI. To determine the most effective condition for dissociation of coupled subtilisin BPN', effects of pH, ionic strength, protein denaturants, and sodium dodecyl sulfate (SDS) were examined. Dissociated subtilisin BPN' with high specific activity was obtained when SDS was used as dissociating agent and was removed with Dowex 2-X10 column from dissociated enzyme solution. S-SI-Sepharose was applied to purifications of B. subtilis SO4 alkaline protease and α-chymotrypsin, and purified enzymes with high specific activity were obtained.
