Microbial surface display of glucose dehydrogenase for amperometric glucose biosensor.

A genetically engineered Escherichia coli (E. coli) strain displaying glucose dehydrogenase (GDH) with ice-nucleation protein (INP) as the anchoring motif was first constructed. The surface localization and functionality of the fusion protein containing GDH were verified by SDS-PAGE, Western blotting and enzymatic activity assay. The fusion of INP had no effects on the functionality of GDH cofactor binding domain. The activity assay showed that 74.6% of the cell lysate GDH activity was detected in the outer membrane fractions. Compared with the crude enzyme solution from E. coli expressing intracellular GDH, the GDH-displaying bacteria (GDH-bacteria) was stable within pH 6-10 below 40°C. Further, a novel electrochemical glucose biosensor was developed by construction of Nafion/GDH-bacteria/multiwalled-carbon-nanotube modified electrode. The as-prepared biosensor is linear with the concentration of d-glucose within the range of 50-800 μM and a low detection limit of 4 μM D-glucose (S/N=3). Excess saccharides including D-galactose, D-fructose, D-cellbiose, L-arabinose and D-sucrose, D-maltose, D-mannose and D-xylose as well as common interfering substances (acetaminophen, ascorbic acid and uric acid) did not affect the detection of D-glucose (0.1mM). The proposed biosensor is stable, specific, reproducible, simple, rapid and cost-effective, which can be used for detection of real samples. It is envisioned that this GDH-bacteria will be found promising applications in biofuel cell, glucose detection and cofactor reproduction system.