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

Qingye Han,Binglian Wang,Zhenyu Zhang,Lushan Wang,Qingai Liu,Weili Gong,Lei Cai,Yiwei Li,Lan Zheng,Qingjun Meng,Yaohong Ma,Yan Yang

doi:10.3390/ijms22115529

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.

Highlights

As an important substance in the management of diabetes and control of food quality and the fermentation process, glucose levels need to be frequently monitored [1]
In the genome of A. niger An76 sequenced by our lab, we found a novel flavin adenine dinucleotide (FAD)-dependent glucose 1-dehydrogenase (FAD-glucose dehydrogenase (GDH)) gene that was phylogenetically distantly related with other FAD-GDHs from Aspergillus species, as shown in Figure 1a, and the sequence identity was only 53.03% with that of the Aspergillus flavus FAD-GDH (AfGDH, PDB ID: 4YNT) [20] (Figure 1b), which has been widely used for commercial self-monitoring of blood glucose sensors
The GDH-natural linker (NL)-carbohydrate binding module family 2 (CBM2) encoding gene was constructed by inserting the NL and CBM2 gene between the GDH and His tag encoding gene

Summary

Introduction

As an important substance in the management of diabetes and control of food quality and the fermentation process, glucose levels need to be frequently monitored [1]. The reactions catalyzed by GOx are affected by the dissolved O2 concentration; if the detection is based on measuring the H2 O2 level, the electrode surface reactions are affected by the applied, extreme potential (usually over +600 mV vs standard electrode), which opens up the sensor system for interfering reactions and causes significant bias in the measurement [3] To circumvent this problem, alternative enzymes, especially glucose dehydrogenase (GDH), which are insensitive to oxygen and use artificial redox mediators with a lower potential range to shuttle electrons from the enzyme to the electrode, have become attractive for biosensors [4]

Methods

Results

Conclusion