title: Microbial surface display of glucose dehydrogenase for amperometric glucose biosensor.
abstract: 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.
authors: Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China.
authors: Bo
authors: B
authors: Liang
authors: Liang
authors: L
authors: Li
authors: Xiangjiang
authors: X
authors: Tang
authors: Qiaolin
authors: Q
authors: Lang
authors: Hongwei
authors: H
authors: Wang
authors: Feng
authors: F
authors: Li
authors: Jianguo
authors: J
authors: Shi
authors: Wei
authors: W
authors: Shen
authors: Ilaria
authors: I
authors: Palchetti
authors: Marco
authors: M
authors: Mascini
authors: Aihua
authors: A
authors: Liu
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D004563:Electrochemistry; D004566:Electrodes; D004926:Escherichia coli / Q000201:enzymology; D005947:Glucose / Q000302:isolation & purification*; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry*; D006801:Humans; D057230:Limit of Detection; D037742:Nanotubes, Carbon / Q000737:chemistry; D010084:Oxidation-Reduction
chemical_list: D001425:Bacterial Outer Membrane Proteins; D037742:Nanotubes, Carbon; C063113:ice nucleation protein; D042843:Glucose 1-Dehydrogenase; D005947:Glucose