A COLORIMETRIC ASSAY FOR PENICILLIN-V AMIDASE

The hydrolysis of penicillin-V to phenoxyacetic acid and 6-aminopenicillanic acid by the fungal enzyme penicillin-V amidase is of industrial importance since the 6-aminopenicillanic acid produced is an intermediate for semisynthetic penicillins. A rapid colorimetric assay of penicillin-V amidase was developed which uses 2-nitro-5-(phenoxyacetamido)-benzoic acid as a substrate. The released chromophore, 2-amino-5-nitrobenzoic acid, was detected at 405 nm. Using penicillin V amidase from the fungus Fusarium oxysporum, the K(M) and V(max) for this substrate were 0.89 mM and 2.6 μmol/min/mg enzyme, respectively. Hydrolysis could be competitively inhibited by penicillin-V with a K(i) of 4 mM. The change in the initial velocity of hydrolysis of 2-nitro-5-(phenoxyacetamido)-benzoic acid at 500 μM was linear over the range of 0.5 to 10 μg/ml enzyme. These results show that this new compound is useful in determining the presence and levels of penicillin-V amidase. © 1994 Academic Press, Inc. All rights reserved.