Cell bound and extracellular glucose Oxidases from Aspergillus niger BTL:: Evidence for a secondary glycosylation mechanism

Two glucose oxidase (GOX) isoforms where purified to electrophoretic homogeneity from the mycelium extract (GOX(I)) and the extracellular medium (GOX(II)) of Aspergillus niger BTL cultures. Both enzymes were found to be homodimers with nonreduced molecular masses of 148 and 159 kDa and pI values of 3.7 and 3.6 for GOX(I) and GOX(II), respectively. The substrate specificity and the kinetic characteristics of the two GOX forms, as expressed through their apparent Km values on glucose, as well as pH and T activity optima, were almost identical. The only structural difference between the two enzymes was in their degrees of glycosylation, which were determined equal to 14.1 and 20.8% (w/w) of their molecular masses for GOX(I) and GOX(II), respectively. The above difference in the carbohydrate content between the two enzymes seems to influence their pH and thermal stabilities. GOX(II) proved to be more stable than GOX(I) at pH values 2.5, 3.0, 8.0, and 9.0. Half-lives of GOX(I) at pH 3.0 and 8.0 were 8.9 and 17.5 h, respectively, whereas the corresponding values for GOX(II) were 13.5 and 28.1 h. As far as the thermal stability is concerned, GOX(II) was also more thermostable than GOX(I) as judged by the deactivation constants determined at various temperatures. More specifically, the half-lives of GOX(I) and GOX(II), at 45 C, were 12 and 49 h, respectively. These results suggest A. niger BTL probably possesses a secondary glycosylation mechanism that increases the stability of the excreted GOX.