Abstract
The culture supernatants of Clostridium botulinum A, B, and F types (all proteolytic) contained two different enzymes. One of the enzymes had esterase activity against BAEE (α-N-benzoyl-arginine ethyl ester) and the other, proteinase activity against casein. Both enzymes were purified by the ammonium sulfate precipitation, DEAE-Sephadex chromatography, and gel filtration. These proteinases showed maximal activity at pH 9 against casein and were not inhibited by 1×10-3M DFP. These proteinases hydrolyzed di-and tripeptides, such as DL-alanylglycine, DL-alanylglycylglycine, DL-leucylglycine, and DL-leucylglycylglycine, but exclusive of glycylglycine and glycylglycylglycine. The molecular weights of the proteinases obtained from the 3 types were estimated to be about 25, 000 (A and B types) and 35, 000 (F type). The other properties and actions were the same among these enzymes. On the other hand, the purified esterases from the 3 types showed maximal activity at pH 7 against BAEE. The esterase activities were enhanced by the addition of 1×10-3M Ca++, Zn++, Mg++, and KCN. Cu++, cysteine, and PCMB reduced the esterase activity by 40-50%, but DFP did not. The molecular weight of esterase derived from each of the 3 types was estimated to be about 67, 000. The enzymes obtained from the 3 types had the same properties and actions.
