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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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Stability tests: a Variation of the response current for glucose with time for GcGDH/Os-polymer and AspGDH/Os-polymer modified electrodes in a constant flow of 5 mM glucose solution throughout the duration of test up to 25 h. Experiments were performed in 50 mM phosphate buffer at pH 7.4. The flow rate was 0.5 mL min−1 and the working potential was set to +175 mV vs. Ag/AgCl0.1 M KCl. b Variation of the response current with time for GcGDH/Os-polymer modified electrode with consecutive injections of 5 mM glucose as substrate for 6 days. Experiments were performed in 50 mM phosphate buffer at pH 7.4. The flow rate was 0.5 mL min−1 and the applied potential was set to +175 mV vs. Ag/AgCl0.1 M KCl
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Fig3: Stability tests: a Variation of the response current for glucose with time for GcGDH/Os-polymer and AspGDH/Os-polymer modified electrodes in a constant flow of 5 mM glucose solution throughout the duration of test up to 25 h. Experiments were performed in 50 mM phosphate buffer at pH 7.4. The flow rate was 0.5 mL min−1 and the working potential was set to +175 mV vs. Ag/AgCl0.1 M KCl. b Variation of the response current with time for GcGDH/Os-polymer modified electrode with consecutive injections of 5 mM glucose as substrate for 6 days. Experiments were performed in 50 mM phosphate buffer at pH 7.4. The flow rate was 0.5 mL min−1 and the applied potential was set to +175 mV vs. Ag/AgCl0.1 M KCl

Mentions: The operational stability is one of the key parameters of a biosensor. The long-term stability is not only beneficial to biosensor transport and storage but also helps to decrease the per measurement costs [55]. The operational stabilities of the GcGDH/Os-polymer and the AspGDH/Os-polymer modified electrodes were investigated using a constant flow of a 5 mM glucose solution throughout the duration of the test. Figure 3a shows the results of these stability tests performed for 25 h. It should be noted that there was a slow decrease in the current response of both modified electrodes in the first 15 h, which further decreased for the AspGDH/Os-polymer-based electrode, but less strongly for the GcGDH/Os-polymer-based electrode. After 25 h with a constant flow of 5 mM glucose, both modified electrodes kept almost 40% of their initial response to glucose.Fig. 3


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)

Stability tests: a Variation of the response current for glucose with time for GcGDH/Os-polymer and AspGDH/Os-polymer modified electrodes in a constant flow of 5 mM glucose solution throughout the duration of test up to 25 h. Experiments were performed in 50 mM phosphate buffer at pH 7.4. The flow rate was 0.5 mL min−1 and the working potential was set to +175 mV vs. Ag/AgCl0.1 M KCl. b Variation of the response current with time for GcGDH/Os-polymer modified electrode with consecutive injections of 5 mM glucose as substrate for 6 days. Experiments were performed in 50 mM phosphate buffer at pH 7.4. The flow rate was 0.5 mL min−1 and the applied potential was set to +175 mV vs. Ag/AgCl0.1 M KCl
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3275720&req=5

Fig3: Stability tests: a Variation of the response current for glucose with time for GcGDH/Os-polymer and AspGDH/Os-polymer modified electrodes in a constant flow of 5 mM glucose solution throughout the duration of test up to 25 h. Experiments were performed in 50 mM phosphate buffer at pH 7.4. The flow rate was 0.5 mL min−1 and the working potential was set to +175 mV vs. Ag/AgCl0.1 M KCl. b Variation of the response current with time for GcGDH/Os-polymer modified electrode with consecutive injections of 5 mM glucose as substrate for 6 days. Experiments were performed in 50 mM phosphate buffer at pH 7.4. The flow rate was 0.5 mL min−1 and the applied potential was set to +175 mV vs. Ag/AgCl0.1 M KCl
Mentions: The operational stability is one of the key parameters of a biosensor. The long-term stability is not only beneficial to biosensor transport and storage but also helps to decrease the per measurement costs [55]. The operational stabilities of the GcGDH/Os-polymer and the AspGDH/Os-polymer modified electrodes were investigated using a constant flow of a 5 mM glucose solution throughout the duration of the test. Figure 3a shows the results of these stability tests performed for 25 h. It should be noted that there was a slow decrease in the current response of both modified electrodes in the first 15 h, which further decreased for the AspGDH/Os-polymer-based electrode, but less strongly for the GcGDH/Os-polymer-based electrode. After 25 h with a constant flow of 5 mM glucose, both modified electrodes kept almost 40% of their initial response to glucose.Fig. 3

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