High-level secretory expression of penicillin amidase from Providencia rettgeri in Saccharomyces cerevisiae: purification and characterization.
Heterologous production of the heterodimeric penicillin G amidase (PAC) from Providencia rettgeri was optimized in Saccharomyces cerevisiae. 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 rPAC(Pr), 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 PAC(Pr) was further characterized in terms of specific activity, pH, and temperature profiles and kinetic parameters. The data presented here suggest that by overexpressing rPAC(Pr) in S.cerevisiae 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.
Ljubijankić
Goran
G
Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, 11001 Belgrade, FR Yugoslavia, Italy. ljubijan@EUnet.yu
Gvozdenović
Jelena
J
Sevo
Milica
M
Degrassi
Giuliano
G
D019149:Bioreactors; D003470:Culture Media / Q000494:pharmacology; D004274:DNA, Recombinant / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D010100:Oxygen / Q000378:metabolism; D010399:Penicillin Amidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011532:Providencia / Q000737:chemistry* / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology*; D013696:Temperature; D013997:Time Factors
D003470:Culture Media; D004274:DNA, Recombinant; D010399:Penicillin Amidase; D010100:Oxygen