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

Int J Mol Sci. 2021 May 24;22(11):5529. doi: 10.3390/ijms22115529.


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.

Keywords: FAD-dependent glucose 1-dehydrogenase; biosensor; carbohydrate-binding module family 2 tag; nanocellulose; orientated immobilization.

MeSH terms

  • Animals
  • Aspergillus flavus / chemistry
  • Aspergillus flavus / metabolism
  • Biosensing Techniques / instrumentation
  • Biosensing Techniques / methods*
  • Blood Glucose / analysis
  • Electrodes
  • Enzyme Assays / methods*
  • Enzymes, Immobilized / chemistry
  • Enzymes, Immobilized / metabolism*
  • Escherichia coli / metabolism
  • Flavin-Adenine Dinucleotide / metabolism*
  • Fungi / chemistry
  • Gene Expression
  • Glucose / analysis*
  • Glucose 1-Dehydrogenase / chemistry
  • Glucose 1-Dehydrogenase / genetics
  • Glucose 1-Dehydrogenase / metabolism*
  • Hydrogen-Ion Concentration
  • Microscopy, Electron, Scanning
  • Phylogeny
  • Rats
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Reproducibility of Results
  • Sequence Alignment
  • Temperature


  • Blood Glucose
  • Enzymes, Immobilized
  • Recombinant Proteins
  • Flavin-Adenine Dinucleotide
  • Glucose 1-Dehydrogenase
  • Glucose