Purification and properties of penicillin amidase from Bacillus megaterium.

A penicillin amidase, obtained from the exogenous medium of a Bacillus megaterium culture, was purified approximately 96-fold by means of two cycles of adsorption on, and elution from, Celite, followed by a further fractionation on carboxymethylcellulose. On the basis of sedimentation centrifugation analysis, the final preparation was deemed to be homogeneous with an apparent molecular weight of approximately 120,000. The enzyme is specific for benzylpenicillin and has a pH optimum between 8 and 9. Complete hydrolysis of benzylpenicillin was obtained at low substrate concentrations. At higher substrate concentrations, the hydrolysis of benzylpenicillin was incomplete, apparently due to enzyme inhibition by phenylacetic acid and 6-aminopenicillanic acid, which were formed during the hydrolysis. Under the assay conditions, phenylacetic acid was a competitive inhibitor of penicillin amidase with an inhibitor constant (K(i)) of 0.45 m, whereas 6-aminopenicillanic acid was noncompetitive in nature with a K(i) of 2.6 x 10(-2)m. The Michaelis constant of this enzyme was found to be 4.5 x 10(-3)m when benzylpenicillin was used as substrate.