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 KM and Vmax for this substrate were 0.89 mM and 2.6 mumol/min/mg enzyme, respectively. Hydrolysis could be competitively inhibited by penicillin-V with a Ki of 4 mM. The change in the initial velocity of hydrolysis of 2-nitro-5-(phenoxyacetamido)-benzoic acid at 500 microM was linear over the range of 0.5 to 10 micrograms/ml enzyme. These results show that this new compound is useful in determining the presence and levels of penicillin-V amidase.
Kerr
D E
DE
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121.
D003124:Colorimetry; D006868:Hydrolysis; D007700:Kinetics; D010399:Penicillin Amidase / Q000032:analysis* / Q000378:metabolism; D010404:Penicillin V / Q000032:analysis* / Q000378:metabolism; D010642:Phenoxyacetates / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D010642:Phenoxyacetates; D010399:Penicillin Amidase; D010404:Penicillin V