Localization of cell-bound penicillinase in Bacillus licheniformis.

When protoplasts are prepared from Bacillus licheniformis (strain 749/C, constitutive for penicillinase), approximately 60% of the cell-bound penicillinase is released. The remainder is retained by the protoplast and cannot be removed by washing. This release is specific, in that less than 7% of the cellular reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase and alpha-glucosidase is liberated by the treatment. The freed penicillinase is excluded from G-200 Sephadex, and it is partially sedimented with a force of 65,000 x g for 20 hr. It is probably attached to characteristic tubular and vesicular structures with single-layered membranes that are comparable to structures previously described in intact penicillinase-forming cells. The specific activity of the organelle is more than six times that of twice washed peripheral membrane; furthermore, about 8% of the protein of the structure is penicillinase. At substrate concentrations (benzylpenicillin) of about one-fifth the K(m) value, whole cells show a slight permeability restriction, although this does not occur in isolated particles and protoplasts.