A coupled enzyme assay for isopenicillin N synthetase.
The development of a coupled enzyme assay for the determination of isopenicillin N synthetase activity in purified extracts from Cephalosporium acremonium was described. Isopenicillin N formed from its precursor, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), by the synthetase was hydrolyzed by beta-lactamase I to the corresponding penicilloic acid. Automatic titration of the acid with standard sodium hydroxide delivered by a pH-stat gave a continuous plot of product formed vs time. This assay has been used in kinetic studies and to determine the effects of pH, ionic strength, and temperature on the enzyme's activity.
D000164:Acremonium / Q000201:enzymology; D002384:Catalysis; D004798:Enzymes / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009994:Osmolar Concentration; D010088:Oxidoreductases; D013696:Temperature; D001618:beta-Lactamases
D004798:Enzymes; D010088:Oxidoreductases; C038370:isopenicillin N synthetase; D001618:beta-Lactamases