Orientated Immobilization of FAD-Dependent Glucose Dehydrogenase on Electrode by Carbohydrate-Binding Module Fusion for Efficient Glucose Assay

Abstract

The discovery or engineering of fungus-derived FAD-dependent glucose 1-dehydrogenase (FAD-GDH) is especially important in the fabrication and performance of glucose biosensors. In this study, a novel FAD-GDH gene, phylogenetically distantly with other FAD-GDHs from Aspergillus species, was identified. Additionally, the wild-type GDH enzyme, and its fusion enzyme (GDH-NL-CBM2) with a carbohydrate binding module family 2 (CBM2) tag attached by a natural linker (NL), were successfully heterogeneously expressed. In addition, while the GDH was randomly immobilized on the electrode by conventional methods, the GDH-NL-CBM2 was orientationally immobilized on the nanocellulose-modified electrode by the CBM2 affinity adsorption tag through a simple one-step approach. A comparison of the performance of the two electrodes demonstrated that both electrodes responded linearly to glucose in the range of 0.12 to 40.7 mM with a coefficient of determination R2 > 0.999, but the sensitivity of immobilized GDH-NL-CBM2 (2.1362 × 10−2 A/(M*cm2)) was about 1-fold higher than that of GDH (1.2067 × 10−2 A/(M*cm2)). Moreover, a lower detection limit (51 µM), better reproducibility (<5%) and stability, and shorter response time (≈18 s) and activation time were observed for the GDH-NL-CBM2-modified electrode. This facile and easy immobilization approach used in the preparation of a GDH biosensor may open up new avenues in the development of high-performance amperometric biosensors.

Enzyme immobilization on α-1,3-glucan: development of flow reactor with fusion protein of α-1,3-glucan binding domains and histamine dehydrogenase

“…One way to improve enzyme immobilization is to construct recombinant enzymes that can easily bind to supports and matrixes (Zhou et al, 2014;Han et al, 2021). In a previous study, we constructed cellulose binding domain-fused FMN-dependent NADHazoreductase (AZR-CBD) and glucose 1-dehydrogenase (CBD-GDH) (Yano et al, 2019), and both enzymes retained their catalytic activity after immobilization onto cellulose.…”