Structural and biochemical properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense |
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Authors: | H M Kalisz J Hendle R D Schmid |
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Institution: | (1) GBF – Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124 Braunschweig, Germany Tel.: +49 531 6181305 Fax.: +49 531 6181302 e-mail: kalisz@gbf-braunschweig.de, DE |
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Abstract: | 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 °C and pH 7–8, and 2 M KF, which stabilised the enzyme up to 36-fold
at 60 °C and pH 6. In sodium acetate buffer, changes in pH (4–6) affected the affinity for glucose but had no effect on the
V
max of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in V
max and a 2-fold decrease in K
m were observed.
Received: 8 October 1996 / Received revision: 14 January 1997 / Accepted: 17 January 1997 |
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