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Characterization of different FAD-dependent glucose dehydrogenases for possible use in glucose-based biosensors and biofuel cells.

Zafar MN, Beden N, Leech D, Sygmund C, Ludwig R, Gorton L - Anal Bioanal Chem (2012)

Bottom Line: One tested FADGDH was that recently discovered in Glomerella cingulata (GcGDH), another was the recombinant form expressed in Pichia pastoris (rGcGDH), and the third was a commercially available glycosylated enzyme from Aspergillus sp. (AspGDH).Additionally, deglycosylated rGcGDH (dgrGcGDH) was investigated to see whether the reduced glycosylation would have an effect, e.g., a higher current density, which was indeed found.GcGDH/Os-polymer modified electrodes were also used and investigated for their selectivity for a number of different sugars.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Structural Biology, Lund University, Lund, Sweden. MNadeem.Zafar@biochemistry.lu.se

ABSTRACT
In this study, different flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) were characterized electrochemically after "wiring" them with an osmium redox polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)(10)Cl](+) on graphite electrodes. One tested FADGDH was that recently discovered in Glomerella cingulata (GcGDH), another was the recombinant form expressed in Pichia pastoris (rGcGDH), and the third was a commercially available glycosylated enzyme from Aspergillus sp. (AspGDH). The performance of the Os-polymer "wired" GDHs on graphite electrodes was tested with glucose as the substrate. Optimal operational conditions and analytical characteristics like sensitivity, linear ranges and current density of the different FADGDHs were determined. The performance of all three types of FADGDHs was studied at physiological conditions (pH 7.4). The current densities measured at a 20 mM glucose concentration were 494 ± 17, 370 ± 24, and 389 ± 19 μA cm(-2) for GcGDH, rGcGDH, and AspGDH, respectively. The sensitivities towards glucose were 2.16, 1.90, and 1.42 μA mM(-1) for GcGDH, rGcGDH, and AspGDH, respectively. Additionally, deglycosylated rGcGDH (dgrGcGDH) was investigated to see whether the reduced glycosylation would have an effect, e.g., a higher current density, which was indeed found. GcGDH/Os-polymer modified electrodes were also used and investigated for their selectivity for a number of different sugars.

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A Effect of pH on the response of the GcGDH/Os-polymer, rGcGDH/Os-polymer and AspGDH/Os-polymer modified graphite electrodes. Experiments were performed in 50 mM phosphate buffer containing 20 mM glucose solution. The flow rate was 0.5 mL min−1 and the working potential was set to +175 vs. Ag/AgCl0.1 M KCl. B Calibration graphs (current densities) of GcGDH/Os-polymer, rGcGDH/Os-polymer and AspGDH/Os-polymer modified graphite electrodes. Experiments were performed in 50 mM phosphate buffer at pH 7.4 containing various glucose concentrations. The flow rate was 0.5 mL min−1 and the working potential was set to +175 mV vs. Ag/AgCl0.1 M KCl. C Linear range of the glucose calibration curves of GcGDH/Os-polymer, rGcGDH/Os-polymer and AspGDH/Os-polymer modified graphite
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Fig2: A Effect of pH on the response of the GcGDH/Os-polymer, rGcGDH/Os-polymer and AspGDH/Os-polymer modified graphite electrodes. Experiments were performed in 50 mM phosphate buffer containing 20 mM glucose solution. The flow rate was 0.5 mL min−1 and the working potential was set to +175 vs. Ag/AgCl0.1 M KCl. B Calibration graphs (current densities) of GcGDH/Os-polymer, rGcGDH/Os-polymer and AspGDH/Os-polymer modified graphite electrodes. Experiments were performed in 50 mM phosphate buffer at pH 7.4 containing various glucose concentrations. The flow rate was 0.5 mL min−1 and the working potential was set to +175 mV vs. Ag/AgCl0.1 M KCl. C Linear range of the glucose calibration curves of GcGDH/Os-polymer, rGcGDH/Os-polymer and AspGDH/Os-polymer modified graphite

Mentions: The pH dependence of the FADGDH/Os-polymer modified graphite electrodes was tested in the electrochemical flow-through cell by varying the pH of the phosphate carrier buffer solution between 6.0 and 8.5. Figure 2a shows the results obtained by injecting samples of 20 mM glucose. Compared to the other two GDHs, AspGDH exhibits a slightly broader pH profile between pH 7.0–8.0 and therefore also less sensitive to pH drifts of an electrode, but for all three measured GDHs the pH optimum was found to be pH 7.5. At this pH, which is close to the pH of physiological conditions, GcGDH (35.1 μA) showed the highest activity, as revealed by the response current, followed by rGcGDH (31.7 μA), and AspGDH (30.5 μA).Fig. 2


Characterization of different FAD-dependent glucose dehydrogenases for possible use in glucose-based biosensors and biofuel cells.

Zafar MN, Beden N, Leech D, Sygmund C, Ludwig R, Gorton L - Anal Bioanal Chem (2012)

A Effect of pH on the response of the GcGDH/Os-polymer, rGcGDH/Os-polymer and AspGDH/Os-polymer modified graphite electrodes. Experiments were performed in 50 mM phosphate buffer containing 20 mM glucose solution. The flow rate was 0.5 mL min−1 and the working potential was set to +175 vs. Ag/AgCl0.1 M KCl. B Calibration graphs (current densities) of GcGDH/Os-polymer, rGcGDH/Os-polymer and AspGDH/Os-polymer modified graphite electrodes. Experiments were performed in 50 mM phosphate buffer at pH 7.4 containing various glucose concentrations. The flow rate was 0.5 mL min−1 and the working potential was set to +175 mV vs. Ag/AgCl0.1 M KCl. C Linear range of the glucose calibration curves of GcGDH/Os-polymer, rGcGDH/Os-polymer and AspGDH/Os-polymer modified graphite
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3275720&req=5

Fig2: A Effect of pH on the response of the GcGDH/Os-polymer, rGcGDH/Os-polymer and AspGDH/Os-polymer modified graphite electrodes. Experiments were performed in 50 mM phosphate buffer containing 20 mM glucose solution. The flow rate was 0.5 mL min−1 and the working potential was set to +175 vs. Ag/AgCl0.1 M KCl. B Calibration graphs (current densities) of GcGDH/Os-polymer, rGcGDH/Os-polymer and AspGDH/Os-polymer modified graphite electrodes. Experiments were performed in 50 mM phosphate buffer at pH 7.4 containing various glucose concentrations. The flow rate was 0.5 mL min−1 and the working potential was set to +175 mV vs. Ag/AgCl0.1 M KCl. C Linear range of the glucose calibration curves of GcGDH/Os-polymer, rGcGDH/Os-polymer and AspGDH/Os-polymer modified graphite
Mentions: The pH dependence of the FADGDH/Os-polymer modified graphite electrodes was tested in the electrochemical flow-through cell by varying the pH of the phosphate carrier buffer solution between 6.0 and 8.5. Figure 2a shows the results obtained by injecting samples of 20 mM glucose. Compared to the other two GDHs, AspGDH exhibits a slightly broader pH profile between pH 7.0–8.0 and therefore also less sensitive to pH drifts of an electrode, but for all three measured GDHs the pH optimum was found to be pH 7.5. At this pH, which is close to the pH of physiological conditions, GcGDH (35.1 μA) showed the highest activity, as revealed by the response current, followed by rGcGDH (31.7 μA), and AspGDH (30.5 μA).Fig. 2

Bottom Line: One tested FADGDH was that recently discovered in Glomerella cingulata (GcGDH), another was the recombinant form expressed in Pichia pastoris (rGcGDH), and the third was a commercially available glycosylated enzyme from Aspergillus sp. (AspGDH).Additionally, deglycosylated rGcGDH (dgrGcGDH) was investigated to see whether the reduced glycosylation would have an effect, e.g., a higher current density, which was indeed found.GcGDH/Os-polymer modified electrodes were also used and investigated for their selectivity for a number of different sugars.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Structural Biology, Lund University, Lund, Sweden. MNadeem.Zafar@biochemistry.lu.se

ABSTRACT
In this study, different flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) were characterized electrochemically after "wiring" them with an osmium redox polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)(10)Cl](+) on graphite electrodes. One tested FADGDH was that recently discovered in Glomerella cingulata (GcGDH), another was the recombinant form expressed in Pichia pastoris (rGcGDH), and the third was a commercially available glycosylated enzyme from Aspergillus sp. (AspGDH). The performance of the Os-polymer "wired" GDHs on graphite electrodes was tested with glucose as the substrate. Optimal operational conditions and analytical characteristics like sensitivity, linear ranges and current density of the different FADGDHs were determined. The performance of all three types of FADGDHs was studied at physiological conditions (pH 7.4). The current densities measured at a 20 mM glucose concentration were 494 ± 17, 370 ± 24, and 389 ± 19 μA cm(-2) for GcGDH, rGcGDH, and AspGDH, respectively. The sensitivities towards glucose were 2.16, 1.90, and 1.42 μA mM(-1) for GcGDH, rGcGDH, and AspGDH, respectively. Additionally, deglycosylated rGcGDH (dgrGcGDH) was investigated to see whether the reduced glycosylation would have an effect, e.g., a higher current density, which was indeed found. GcGDH/Os-polymer modified electrodes were also used and investigated for their selectivity for a number of different sugars.

Show MeSH
Related in: MedlinePlus