PURIFICATION AND CHARACTERIZATION OF THE ISOPENICILLIN-N EPIMERASE FROM NOCARDIA-LACTAMDURANS

Isopenicillin N (IPN) epimerase, an enzyme involved in cephalosporin and cephamycin biosynthesis that converts IPN into penicillin N, was extracted from Nocardia lactamdurans and purified 88-fold. The enzyme was unstable but could be partially stabilized by addition of pyridoxal phosphate. The purified enzyme did not require ATP for activity in contrast to other amino acid racemases. The enzyme had an M(r) of 59,000 as determined by gel filtration; IPN epimerase from Streptomyces clavuligerus had an M(r) of 63,000. A protein band of M(r) 59,000 was found to be enriched in SDS-PAGE of active fractions from N. lactamdurans. The optimal temperature of the epimerase was 25°C and the optimal pH 7.0. The apparent K(m) for IPN was 270 μM. Fe2+, Cu2+, Hg2+ and Zn2+ strongly inhibited enzyme activity. α-Aminoadipic acid, valine, glutamine, glycine, aspartic acid and glutathione do not affect enzyme activity, whereas ammonium sulphate was inhibitory. The epimerase activity was partially inhibited by several thiol-specific reagents.