Penicillin acylase from the hybrid strain Escherichia coli 5K(pHM12): Enzyme formation and hydrolysis of β-lactam antibiotics with whole cells

Abstract

Penicillin acylase formation by the hybrid strain Escherichia coli 5K(pHM12) was studied under different culture conditions and reached 200 to 250 μmol of 6-aminopenicillanic acid per min per g of bacteria (wet weight) for penicillin G. The K(m) of whole-cell acylase was determined with 9 to 11 mM for penicillin G at a pH optimum of 7.8 at 45°C. a competitive product inhibition for phenylacetic acid of K(i) = 130 mM was found. 6-Aminopenicillanic acid acts as a noncompetitive inhibitor, with a K(i) of 131. The temperature optimum of the reaction lies at 54°C. Penicillin G inhibits the reaction at K(i)(S) = 1.565 to 1.570 mM. Whole-cell acylase reacts on a wide spectrum of penicillins and cephalosporins, but those substrates with a δ-aminoadipyl rest are not hydrolized. β-Lactamase activity of <1% relative to the acylase actvity was found at reaction temperatures between 28 and 45°C. After a comparison of different methods for the estimation of β-lactamase activity, we found that high-pressure liquid chromatography is to be preferred. During batch fermentation of E. coli 5K(pHM12), problems of plasmid stability in the host strain arose which were overcome by the addition of 4 mg of tetracycline per liter to the medium as a selective marker.