Penicillin acylase from the hybrid strains Escherichia coli 5K(pHM12): enzyme formation and hydrolysis of beta-lactam antibiotics with whole cells.

Penicillin acylase formation by the hybrid strain Escherichia coli 5K(pHM12) was studied under different culture conditions and reached 200 to 250 mumol of 6-aminopenicillanic acid per min per g of bacteria (wet weight) for penicillin G. The Km of whole-cell acylase was determined with 9 to 11 mM for penicillin G at a pH optimum of 7.8 at 45 degrees C. A competitive product inhibition for phenylacetic acid of Ki = 130 mM was found. 6-Aminopenicillanic acid acts as a noncompetitive inhibitor, with a Ki of 131. The temperature optimum of the reaction lies at 54 degrees C. Penicillin G inhibits the reaction at Ki(S) = 1,565 to 1,570 mM. Whole-cell acylase reacts on a wide spectrum of penicillins and cephalosporins, but those substrates with a delta-aminoadipyl rest are not hydrolized. beta-Lactamase activity of less than 1% relative to the acylase activity was found at reaction temperatures between 28 and 45 degrees C. After a comparison of different methods for the estimation of beta-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.