Open AccessProduction of glycosylated thermostable Providencia rettgeri penicillin G amidase in Pichia pastoris
Author(s) -
Ševo Milica,
Degrassi Giuliano,
Skoko Nataša,
Venturi Vittorio,
Ljubijankić Goran
Publication year - 2002
Publication title -
fems yeast research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.991
H-Index - 92
eISSN - 1567-1364
pISSN - 1567-1356
DOI - 10.1111/j.1567-1364.2002.tb00045.x
Subject(s) - biology , microbiology and biotechnology , pichia pastoris , providencia , pichia , penicillin , amidase , bacteria , antibiotics , enterobacteriaceae , biochemistry , recombinant dna , escherichia coli , genetics , gene
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.