Comparison ofPenicillium amagasakienseglucose oxidase purified as glyco- and aglyco-proteins

Abstract

Penicillium amagasakiense secreted glucose oxidase (GOD) with a highly diminished carbohydrate moiety (aglyco-GOD) in the presence of 20 mg/l tunicamycin. To examine enzymatic properties of aglyco-GOD, this and native GOD (glyco-GOD) were purified to homogeneity from the culture medium with or without tunicamycin. The carbohydrate content of aglyco-GOD was 2.9% and the Mr of it and its subunit were 130 000 and 70 000, respectively. On the other hand, those from glyco-GOD were 20.4%, 150 000 and 80 000, respectively. Nonetheless, they showed remarkably similar catalytic properties to each other: Km values of 3.4 and 2.7 mM and Vmax values of 320 and 270 U/mg protein. pH optima 5.5 and 6.0 were observed for aglyco and glyco-GOD, respectively. Isoelectric focusing showed a strikingly similar pattern with each GOD having one major band of pI 4.2 and 2 minor bands between pI 4.3 and 4.5, which implies that microheterogeneity of GOD was not due to heterogeneous glycosylation. Up to 35°C, both enzyme preparations were stable for 30 min while both were considerably inactivated after 30 min at 45°C. Therefore, we conclude that the carbohydrate moiety plays no significant role in the enzymatic function of P. amagasakiense GOD.