High-Level Secretory Expression of Penicillin Amidase from Providencia rettgeri in Saccharomyces cereuisiae: Purification and Characterization

Heterologous production of the heterodimeric penicillin G amidase (PAC) from Providencia rettgeri was optimized in Saccharomyces cereuisiae. Several factors, including the effect of different growth and induction conditions, were identified to be critical for the enzyme overproduction and secretion. The PAC yield was significantly increased by more than 500-fold compared to that obtained in the native bacterium, and the recombinant enzyme was almost entirely secreted. Electrophoretic characterization of the secreted rPACpr, which was purified over 20-fold by a combination of hydrophobic interaction and ion-exchange chromatography, demonstrated a microheterogeneity of the recombinant enzyme. The recombinant PACpr was further characterized in terms of specific activity, pH, and temperature profiles and kinetic parameters. The data presented here suggest that by overexpressing rPACpr in S. cereuisiae and purifying secreted enzyme from culture medium one can readily obtain a large amount of an alternative source of penicillin amidase with properties comparable to that of todays main industrial source of enzyme.