title: Direct electron transfer type disposable sensor strip for glucose sensing employing an engineered FAD glucose dehydrogenase.
abstract: The FAD-dependent glucose dehydrogenase (FADGDH) from Burkholderia cepacia has several attractive features for glucose sensing. However, expanding the application of this enzyme requires improvement of its substrate specificity, especially decreasing its high activity toward maltose. A three-dimensional structural model of the FADGDH catalytic subunit was generated by homology modeling. By comparing the predicted active site with that of glucose oxidase, the two amino acid residues serine 326 and serine 365 were targeted for site-directed mutagenesis. The single mutations that produced the highest glucose specificity were combined, leading to the creation of the S326Q/S365Y double mutant, which was virtually nonreactive to maltose while retaining high glucose dehydrogenase activity. The engineered FADGDH was used to develop a direct electron transfer-type, disposable glucose sensor strip by immobilizing the enzyme complex onto a carbon screen-printed electrode. While the electrode employing wild-type FADGDH provided dangerously flawed results in the presence of maltose, the sensor employing our engineered FADGDH showed a clear glucose concentration-dependent response that was not affected by the presence of maltose.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture & Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yamashita
authors.forename: Stefano
authors.initials: S
authors.lastname: Ferri
authors.forename: Mai Linh
authors.initials: ML
authors.lastname: Huynh
authors.forename: Hitomi
authors.initials: H
authors.lastname: Shimizu
authors.forename: Hideaki
authors.initials: H
authors.lastname: Yamaoka
authors.forename: Koji
authors.initials: K
authors.lastname: Sode
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001234:Aspergillus niger / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques; D001786:Blood Glucose / Q000032:analysis*; D015190:Blood Glucose Self-Monitoring / Q000295:instrumentation* / Q000379:methods; D016956:Burkholderia cepacia / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D003198:Computer Simulation; D055664:Electrochemical Techniques; D004579:Electron Transport; D004926:Escherichia coli; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D042843:Glucose 1-Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D005948:Glucose Dehydrogenases / Q000378:metabolism; D005949:Glucose Oxidase / Q000378:metabolism; D006801:Humans; D008320:Maltose / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011934:Reagent Strips; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D001786:Blood Glucose; D005656:Fungal Proteins; D011934:Reagent Strips; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D008320:Maltose; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase; D005949:Glucose Oxidase; C033955:glucose dehydrogenase (pyrroloquinoline-quinone); D005947:Glucose