MOLECULAR-CLONING AND ANALYSIS OF THE GENE ENCODING THE THERMOSTABLE PENICILLIN-G ACYLASE FROM ALCALIGENES-FAECALIS

Alcaligenes faecalis penicillin G acylase is more stable than the Esch erichia coli enzyme. The activity of the A. faecalis enzyme was not af fected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatm ent. To study the molecular basis of this higher stability, the A. fae calis enzyme was isolated and its gene was cloned and sequenced. The g ene encodes a polypeptide that is characteristic of periplasmic penici llin G acylase (signal peptide-alpha subunit-space-beta subunit). Puri fication, N-terminal amino acid analysis, and molecular mass determina tion of the penicillin G acylase showed that the alpha and beta subuni ts have molecular masses of 23.0 and 62.7 kDa, respectively. The lengt h of the spacer is 37 amino acids. Amino acid sequence alignment demon strated significant homology with the penicillin G acylase from E. col i. A unique feature of the A. faccalis enzyme is the presence of two c ysteines that form a disulfide bridge. The stability of the A. faccali s penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermos tability is attributed to the presence of the disulfide bridge.