Phosphatidylinositol-specific phospholipases C from Bacillus cereus and Bacillus thuringiensis

Publisher Summary Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus and Bacillus thuringiensis catalyzes the cleavage of the sn -3 phosphodiester bond of phosphatidylinositol (PI). Bacillus cereus and B. thuringiensis are closely related rod-shaped gram-positive bacteria that are nonpathogenic to man. The enzymes are excreted in relatively large quantities across the single cell membrane into the growth medium. This facilitates the purification of PI-PLC as intracellular proteins are removed with the cells during an initial centrifugation step. PI-PLC is isolated from the supernatants of B. cereus and B. thuringiensis cultures in milligram quantities. The final product is homogeneous on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), exhibits high specific activity, and is used to raise monoclonal antibodies and provide polypeptide sequence information for the cloning and DNA sequencing of PI-PLC from B. cereus . The B. cereus or B. thuringiensis culture is cooled to 4°, and the cells are removed by centrifugation for 30 min at 13,000 g. Solid ammonium sulfate is added slowly to the supernatant to 90% saturation (576 g/liter), and the solution is kept overnight at 4°. The purity of the final enzyme preparations is generally better than 95%, with similar specific activities for the B. cereus and B. thuringiensis enzymes in the range of 1200 to 1500 units/mg. During the early steps of the purification, the specific enzyme activities are affected by the presence of colored contaminants that contribute strongly in the protein determinations. These contaminants are completely removed by the final Phenyl-Sepharose step.