Isolation and partial purification of a proteolytic enzyme preparation from Clostridium perfringens

Abstract

Although two strains of Clostridium perfringens (ATCC 12915 and 13124) exhibited excellent growth on amino acid and peptone media, only one (ATCC 13124) produced measurable proteolytic enzyme activity. Thus, subsequent purification steps concentrated on isolation of a proteolytic enzyme preparation produced by this strain. Purification and concentration were carried out by precipitating the crude enzyme fraction from the culture filtrate with ZnCl 2, extracting with saturated disodium phosphate and reprecipitating by 60% saturation with (NH 4) 2SO 4. The precipitate was then redissolved in borate buffered saline solution and further purified by successively passing it through a Bio-Gel P-100 column, a DEAE-cellulose column and a Bio-Gel P-200 column. The final step resulted in a 159-fold purification with 12% recovery and a final specific activity of 79 azocoll units/milligramme protein. Although each successive purification step eliminated some of the impurities, the final fraction still showed considerable heterogeneity upon disc-gel electrophoresis.