[54a] Penicillin acylase (assay)

Publisher Summary This chapter presents the assay procedures of penicillin acylase. The most specific method of assaying these enzymes involves the determination of the rate of formation of 6-APA from a readily available penicillin, such as benzylpenicillin or phenoxymethylpenicillin. The method of choice involves the hydroxylamine assay of 6-APA after removal of residual penicillin substrate by solvent extraction at pH 2. Alternatively, the 6-APA may be assayed in the presence of residual substrate by the p-dimethylaminobenzaldehyde method. The method lends itself to automation, but the β-lactam ring opened form of 6-APA also reacts. A non-automated version has also been described. A less specific but readily performed method involves the pH-stat titration of carboxylic acid side chain liberated from the substrate penicillin. This method is suitable for following large-scale reactions or obtaining kinetic data if no β-lactamase enzyme is present to liberate penicilloic acid.