isolation of a penicillin acylase producing e.coli and kinetic characterization of the whole cell enzyme activity

penicillin acylase (ec 3.5.1.11) has been a target of study for a long time because of its pivotal role in the deacylation of the penicillin into the 6- aminopenicillanic acid (6-apa) and the side-chain organic acids. this property of penicillin acylase has been exploited commercially for large scale production of 6-apa, which is the key intermediate in the manufacture of semi-synthetic penicillins. due to the worldwide demand for semi-synthetic penicillins, production of 6-apa has been increased up to 7000 tons in recent years. in this study, sixty-five strains of e. coli were investigated for penicillin acylase activity using fluorescamine method. the 6-aminopenicillanic acid formed in the reaction mixture was developed on thin layer chromatography. one-minute beta-lactamase test was carried out to follow any trace of penicillinase activity. only one sample designated as e.coli ppa78 was found to be penicillin acylase producer. the optimal ph and temperature of penicillin acylase activity of the whole cells were determined to be 8.0 and 57° c, respectively. km value and activation energy of the enzymatic hydrolysis reaction of penicillin g by intracellular enzyme were estimated as 0.004 mmol and 6.2 kcal/mol, respectively.