Properties of Penicillinase from Bacillus cereus 569

Penicillinase from Bacillus cereus 569 was purified and examined for homogeneity by sedimentation analysis, amino-terminal analysis, and vertical acrylamide gel electrophoresis. The enzyme preparation was shown to contain three distinct species of extracellular penicillinase which could be separated easily by gel electrophoresis. Kinetic studies failed to reveal any significant differences in the three species of enzyme. The evidence provided suggests that all three species are composed of a single polypeptide chain of approximately 31,000 molecular weight. The amino acid composition of a mixture of the three species was determined. These data, plus additional experiments, confirmed a lack of cysteine in penicillinase isolated from B. cereus 569. Optical rotatory dispersion data obtained with penicillinase indicate that possibly 30% of the amino acid residues exist in the α helical configuration. Similarly, data obtained with penicillinase reveal that the majority of this α helix is destroyed in the presence of 7.5 m urea or 5.8 m guanidine hydrochloride. These experiments, in conjunction with measurements which show at least a 500-fold reduction in enzyme activity in the presence of the denaturants, indicate that penicillinase from B. cereus 569 is denatured when treated with high molar concentrations of urea or guanidine hydrochloride. Removal of the denaturants results in full restoration of activity and rotatory dispersion very similar to those obtained for the native enzyme. These data, colleclectively, prove that penicillinase from B. cereus 569 can readily undergo reversible denaturation.