Purification and characterization of the thermophilic D-alanine carboxypeptidase from membranes of Bacillus stearothermophilus.

The D-alanine carboxypeptidase, the major penicillinbinding component in membranes of Bacillus stearothermophilus, was purified by covalent a5nity chromatography. The enzyme was solubilized in the nonionic detergent Triton X-100, bound to a column of a 6-aminopenicillanic acidSepharose derivative, and eluted upon treatment of the column with neutral hydroxylamine. The enzyme was thermophilic, possessing a temperature optimum of 55”. Its molecular weight, 46,500, resembled that of the D-alanine carboxypeptidase from Bacillus subtilis. In addition to its carboxypeptidase activity, the enzyme was also capable of carrying out a simple transpeptidation reaction. This reaction, which was penicillin sensitive, involved UDP-N-acetyl mummy1 L alanyl D glutamyl mesodiaminopimelyl D alanyl-D-alanine as transpeptidation donor and either glycine or D-alanine as transpeptidation acceptor. The enzyme, however, did not appear to be the lethal target for P-lactam antibiotics in this organism since the enzyme was 2’7,000-fold more resistant to cephafothin than was the organism.