Fungal FAD-dependent glucose dehydrogenases (FADGDHs) are considered to be superior enzymes for glucose sensor strips because of their insensitivity to oxygen and maltose. One highly desirable mediator for enzyme sensor strips is hexaammineruthenium(III) chloride because of its low redox potential and high storage stability. However, in contrast to glucose oxidase (GOx), fungal FADGDH cannot utilize hexaammineruthenium(III) as electron acceptor. Based on strategic structure comparison between FADGDH and GOx, we constructed a mutant of Aspergillus flavus-derived FADGDH, capable of utilizing hexaammineruthenium(III) as electron acceptor: AfGDH-H403D. In AfGDH-H403D, a negative charge introduced at the pathway-entrance leading to the FAD attracts the positively charged hexaammineruthenium(III) and guides it into the pathway. The corresponding amino acid in wild-type GOx is negatively charged, which explains the ability of GOx to utilize hexaammineruthenium(III) as electron acceptor. Electrochemical measurements showed a response current of 46.0 μA for 10 mM glucose with AfGDH-H403D and hexaammineruthenium(III), similar to that with wild-type AfGDH and ferricyanide (47.8 μA). Therefore, AfGDH-H403D is suitable for constructing enzyme electrode strips with hexaammineruthenium(III) chloride as sole mediator. Utilization of this new, improved fungal FADGDH should lead to the development of sensor strips for blood glucose monitoring with increased accuracy and less stringent packing requirements.

真菌FAD依赖性葡萄糖脱氢酶（FADGDHs）被认为是葡萄糖传感器条的优良酶，因为它们对氧气和麦芽糖不敏感。用于酶传感器条的一种非常理想的介体是六胺六氯化钌（III），因为它的氧化还原电势低且存储稳定性高。但是，与葡萄糖氧化酶（GOx）相比，真菌FADGDH不能利用六氨合钌（III）作为电子受体。基于FADGDH和GOx之间的战略结构比较，我们构建了一个由黄曲霉衍生的FADGDH的突变体，该突变体能够利用六氨合钌（III）作为电子受体：Af GDH-H403D。在非洲GDH-H403D是通向FAD的通道入口处引入的负电荷，它吸引带正电荷的六氨合钌（III）并将其引导至通道中。野生型GOx中相应的氨基酸带负电荷，这解释了GOx利用六氨合钌（III）作为电子受体的能力。电化学测量显示，使用Af GDH-H403D和六氨合钌（III）对10 mM葡萄糖的响应电流为46.0μA，类似于野生型Af GDH和铁氰化物（47.8μA）的响应电流。因此，AfGDH-H403D适用于构建以氯化六氨合钌（III）作为唯一介体的酶电极条。利用这种新的，改良的真菌FADGDH应该可以开发出用于血糖监测的传感器条，其准确性更高且包装要求不那么严格。