Purification and Characteristics of Plasma Membrane Penicillinase from Bacillus licheniformis 749 / C

The plasma membrane-bound penicillinase of Bacillus licheniformis 749/C has been purified from bacteria grown in a medium containing [2-3H]glycerol and 14C-labeled aminoacids, and the purified enzyme compared with exopenicillinase. The procedure consisted of repeated chromatography on DEAE-Sephadex in the presence of Triton X-100, and gel filtration on Sephadex G-75 in the presence of taurodeoxycholate. The purified membrane enzyme moved as a single band in sodium dodecyl sulfate acrylamide gel electrophoresis, and its specific penicillinase activity was 354 units per µg of enzyme protein (equivalent to that of the exoenzyme). The enzyme has an apparent molecular weight of 48,000 in the presence of taurodeoxycholate, as estimated by gel filtration, and exhibited high susceptibility to inactivation by heating and by iodine. The purified enzyme retained characteristic lipophilic properties and contained 3H activity which could not be removed from the enzyme protein by treatment with 8 m urea, 0.2% sodium dodecyl sulfate at 80° or by extraction with chloroform-methanol. The tritium activity could be separated from the enzyme protein by treatment with trypsin or phospholipase D followed by gel filtration and acrylamide gel electrophoresis. The small component(s) released by trypsin contained both 14C and 3H activity. The resulting enzyme had lost its lipophilic character and showed properties similar to exopenicillinase.