title: Production of glycosylated thermostable Providencia rettgeri penicillin G amidase in Pichia pastoris.
abstract: Penicillin G amidase from Providencia rettgeri is a heterodimer of 92 kDa. We have previously expressed the Pr. rettgeri pac gene coding for this enzyme in Saccharomyces cerevisiae, and now we report the expression and characterization in the methylotrophic yeast Pichia pastoris. The recombinant catalytically active enzyme (rPAC(Pr)) was secreted from shake flask-grown P. pastoris cells into the medium at a level of approximately 0.18 U ml(-1). This yield of rPAC(Pr) was higher, by two orders of magnitude, than that obtained using a single-copy expression plasmid in S. cerevisiae. In addition, the secreted recombinant enzyme was entirely N-glycosylated. The recombinant PAC(Pr) was further characterized in terms of specific activity, kinetic parameters and thermostability. Except the significantly higher thermostability of the glycosylated rPAC(Pr) produced in P. pastoris, the other parameters were very similar to those of the corresponding non-glycosylated enzymes produced in bacteria or in S. cerevisiae. The higher thermostability of this recombinant enzyme has a clear industrial advantage.
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, Belgrade, FR Yugoslavia.
authors.forename: Milica
authors.initials: M
authors.lastname: Sevo
authors.forename: Giuliano
authors.initials: G
authors.lastname: Degrassi
authors.forename: Natasa
authors.initials: N
authors.lastname: Skoko
authors.forename: Vittorio
authors.initials: V
authors.lastname: Venturi
authors.forename: Goran
authors.initials: G
authors.lastname: Ljubijankić
mesh_terms: D004795:Enzyme Stability; D006031:Glycosylation; D006358:Hot Temperature; D007700:Kinetics; D010399:Penicillin Amidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D011532:Providencia / Q000201:enzymology* / Q000235:genetics
chemical_list: D010399:Penicillin Amidase