Structural and kinetic properties of nonglycosylated recombinant Penicillium amagasakiense glucose oxidase expressed in Escherichia coli.
The gene coding for Penicillium amagasakiense glucose oxidase (GOX; beta-D-glucose; oxygen 1-oxidoreductase [EC 1.1.3.4]) has been cloned by PCR amplification with genomic DNA as template with oligonucleotide probes derived from amino acid sequences of N- and C-terminal peptide fragments of the enzyme. Recombinant Escherichia coli expression plasmids have been constructed from the heat-induced pCYTEXP1 expression vector containing the mature GOX coding sequence. When transformed into E. coli TG2, the plasmid directed the synthesis of 0.25 mg of protein in insoluble inclusion bodies per ml of E. coli culture containing more than 60% inactive GOX. Enzyme activity was reconstituted by treatment with 8 M urea and 30 mM dithiothreitol and subsequent 100-fold dilution to a final protein concentration of 0.05 to 0.1 mg ml-1 in a buffer containing reduced glutathione-oxidized glutathione, flavin adenine dinucleotide, and glycerol. Reactivation followed first-order kinetics and was optimal at 10 degrees C. The reactivated recombinant GOX was purified to homogeneity by mild acidification and anion-exchange chromatography. Up to 12 mg of active GOX could be purified from a 1-liter E. coli culture. Circular dichroism demonstrated similar conformations for recombinant and native P. amagasakiense GOXs. The purified enzyme has a specific activity of 968 U mg-1 and exhibits kinetics of glucose oxidation similar to those of, but lower pH and thermal stabilities than, native GOX from P. amagasakiense. In contrast to the native enzyme, recombinant GOX is nonglycosylated and contains a single isoform of pI 4.5. This is the first reported expression of a fully active, nonglycosylated form of a eukaryotic, glycosylated GOX in E. coli.
Witt
S
S
GBF-Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Germany.
Singh
M
M
Kalisz
H M
HM
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D005949:Glucose Oxidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D005949:Glucose Oxidase