Purification and properties of the D-alanyl-D-alanine carboxypeptidase of Bacillus coagulans NCIB 9365.

After solubilisation with urea and the non-ionic detergent Genapol X-100, the membrane-bound DD-carboxypeptidase (UDP-N-acetylmuramoyl-tetrapeptidyl-D-alanine alanine-hydrolase, EC 3.4.12.6) of Bacillus coagulans NCIB 9365 was purified to homogeneity, as verified by sodium dodecyl sulphate gel electrophoresis, by chromatography with an ampicillin-agarose affinity resin and DEAE-cellulose. The properties of the purified DD-carboxypeptidase were similar to those of the membrane-bound enzyme; these include enhancement of activity by divalent cations, Pb2+ and Cd2+ being the most effective. The enzyme also catalysed a simple unnatural model transpeptidation reaction between UDP-N-acetylmuramoyl pentapeptide (donor) and D-alanine or glycine (acceptors). The enzyme consisted of a single polypeptide chain with a molecular weight (Mr 29 000), considerably lower than values obtained previously for most other DD-carboxypeptidases. However, its molecular weight and its degree of relatedness, as assessed by amino acid composition, were similar to several beta-lactamases.