Structural and biochemical properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense.
Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety. A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE) for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not-been published for glucose oxidase from P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M (NH4)2SO4, which stabilised glucose oxidase 100- to 300-fold at 50 degrees C and pH 7-8, and 2 M KF, which stabilised the enzyme up to 36-fold at 60 degrees C and pH 6. In sodium acetate buffer, changes in pH (4-6) affected the affinity for glucose but had no effect on the Vmax of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in Vmax and a 2-fold decrease in K(m) were observed.
Kalisz
H M
HM
GBF-Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Germany. kalisz@gbf-braunschweig.de
Hendle
J
J
Schmid
R D
RD
D000595:Amino Acid Sequence; D005949:Glucose Oxidase / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology*
D005949:Glucose Oxidase