ISOLATION OF AN AMINOPEPTIDASE FROM TYPE B CLOSTRIDIUM BOTULINUM

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Abstract
A method for purification of a proteinase found in the culture supernatant of type B C. botulinum (Okra strain) is described. The method involves fractional precipitation with ammonium sulfate and subsequent reprecipitation with sodium chloride under controlled conditions of pH, ammonium sulfate concentration, protein concentration and temperature. This method is rapid and permits a purification of approximately 300-fold with a 15% loss in the amount of enzyme. Solubility measurements and ultracentrifuge analysis of the purified preparation indicate the presence of one component. The proteinase contains little or no tyrosine and tryptophane as shown by its absorption in the UV spectrum. The purified product appears to contain but one proteinase which is activated optimally by Fe++-cysteine; it is an aminopolypeptidase capable of splitting tripeptides but not dipeptides. Fe++ is essential for activity of the enzyme. Binding Fe++ with Versene renders the proteinase inactive.