Extraction of Spore-lytic Enzyme from Clostridium perfringens Spores

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Abstract
Various chemical reagents known to extract spore coat protein were used to extract spore-lytic enzyme (SLE) from intact and germinated spores of C. perfringens. Of the reagents tested, 7.2 M-urea plus 10% (vol/vol) mercaptoethanol, pH 2.85, solubilized the most SLE activity per milligram spores. The quantity of SLE extracted was dependent on the initial pH of the reagent, with a maximum between pH 2.7-3.0. Germinated spores yielded more SLE than non-germinated spores upon urea/mercaptoethanol extraction. SLE release during spore germination probably utilizes a trigger mechanism not satisfied by germination alone. Significant amounts of SLE were released during germination when spores were suspended in potassium chloride or a complex germinant mixture containing brain-heart infusion, yeast extract and chloramphenicol but not during germination with sodium-nitrite, which non-enzymically lysed the cortical peptidoglycan. Greater solubilization of SLE activity was obtained by urea/mercaptoethanol extraction of spores germinated with nitrite than of spores germinated with potassium chloride or the complex germinant.