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 (GOXI) and the extracellular medium (GOXII) 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 GOXI and GOXII, respectively. The substrate specificity and the kinetic characteristics of the two GOX forms, as expressed through their apparent K m 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 GOXI and GOXII, respectively. The above difference in the carbohydrate content between the two enzymes seems to influence their pH and thermal stabilities. GOXII proved to be more stable than GOXI at pH values 2.5, 3.0, 8.0, and 9.0. Half-lives of GOXI at pH 3.0 and 8.0 were 8.9 and 17.5 h, respectively, whereas the corresponding values for GOXII were 13.5 and 28.1 h. As far as the thermal stability is concerned, GOXII was also more thermostable than GOXI as judged by the deactivation constants determined at various temperatures. More specifically, the half-lives of GOXI and GOXII, at 45 degrees 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.
Hatzinikolaou
Dimitris G
DG
Laboratory of Microbiology, Department of Botany, School of Biology, National and Kapodistrian University of Athens, Panepistimioupoli Zografou, Athens, Greece.
Mamma
Diomi
D
Christakopoulos
Paul
P
Kekos
Dimitris
D
D001234:Aspergillus niger / Q000201:enzymology*; D001709:Biotechnology / Q000379:methods*; D002241:Carbohydrates / Q000737:chemistry; D019281:Dimerization; D005947:Glucose / Q000737:chemistry; D005949:Glucose Oxidase / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D025282:Mycelium / Q000737:chemistry; D020033:Protein Isoforms; D013379:Substrate Specificity; D013696:Temperature
D002241:Carbohydrates; D020033:Protein Isoforms; D005949:Glucose Oxidase; D005947:Glucose