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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 Structure of the Os-polymer [Os(4,4′-dimethyl-2,2′-bipyridine)2(PVI)10Cl]+. b Cyclic voltammogram of the Os-polymer on graphite electrode: (solid line) without enzyme, (broken line) with cross-linked GcGDH. Experiments were performed in 50 mM phosphate buffer at pH 7.4 and the scan rate was 10 mV s−1
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Fig1: a Structure of the Os-polymer [Os(4,4′-dimethyl-2,2′-bipyridine)2(PVI)10Cl]+. b Cyclic voltammogram of the Os-polymer on graphite electrode: (solid line) without enzyme, (broken line) with cross-linked GcGDH. Experiments were performed in 50 mM phosphate buffer at pH 7.4 and the scan rate was 10 mV s−1

Mentions: The electrochemical behavior of the Os-polymer [Os(4,4′-dimethyl-2,2′-bipyridine)2(PVI)10Cl]+ was characterized using cyclic voltammetry in the presence and absence of GcGDH in 50 mM potassium phosphate buffer at pH 7.4 without substrate. The structure of this Os-polymer is shown in Fig. 1a. The polymer was selected previously out of six different Os-polymers covering a broad potential range, because it gave the highest current density [37]. The formal redox potentials (E°′) of the polymer was determined to be 21 mV vs. Ag/AgCl0.1 M KCl for [Os(4,4′-dimethyl-2,2′-bipyridine)2(PVI)10Cl]+. This value agrees well with the literature values [40] and with our previous results [14]. The rather low E°′ value minimizes the effects of interfering components by preventing their direct electrochemically oxidation at the electrode surface at high potential. Due to these preliminary results and the fact that the polymer has been already used to successfully “wire” different redox enzymes in biosensors and biofuel cells [14, 46, 47], it was selected for this study. The possibility to successfully “wire” the other FADGDHs to graphite rod electrodes was therefore anticipated [43]. The mechanism of interaction and electron transfer between enzymes and Os-polymers is well described in the following reports [45, 48–50]. The FADGDHs belong to the same structural family of GMC oxidoreductases (glucose-methanol-choline oxidoreductase) as GOx [51]. Similar to GOx, the FADGDHs oxidize glucose at the C1 position and only the β-form is oxidized and 2e−/2H+ are transferred from glucose to the bound FAD, Reaction 1. The electron transfer occurs between FADGDHs and Os-polymers through the following reactions.Fig. 1


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 Structure of the Os-polymer [Os(4,4′-dimethyl-2,2′-bipyridine)2(PVI)10Cl]+. b Cyclic voltammogram of the Os-polymer on graphite electrode: (solid line) without enzyme, (broken line) with cross-linked GcGDH. Experiments were performed in 50 mM phosphate buffer at pH 7.4 and the scan rate was 10 mV s−1
© Copyright Policy
Related In: Results  -  Collection

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

Fig1: a Structure of the Os-polymer [Os(4,4′-dimethyl-2,2′-bipyridine)2(PVI)10Cl]+. b Cyclic voltammogram of the Os-polymer on graphite electrode: (solid line) without enzyme, (broken line) with cross-linked GcGDH. Experiments were performed in 50 mM phosphate buffer at pH 7.4 and the scan rate was 10 mV s−1
Mentions: The electrochemical behavior of the Os-polymer [Os(4,4′-dimethyl-2,2′-bipyridine)2(PVI)10Cl]+ was characterized using cyclic voltammetry in the presence and absence of GcGDH in 50 mM potassium phosphate buffer at pH 7.4 without substrate. The structure of this Os-polymer is shown in Fig. 1a. The polymer was selected previously out of six different Os-polymers covering a broad potential range, because it gave the highest current density [37]. The formal redox potentials (E°′) of the polymer was determined to be 21 mV vs. Ag/AgCl0.1 M KCl for [Os(4,4′-dimethyl-2,2′-bipyridine)2(PVI)10Cl]+. This value agrees well with the literature values [40] and with our previous results [14]. The rather low E°′ value minimizes the effects of interfering components by preventing their direct electrochemically oxidation at the electrode surface at high potential. Due to these preliminary results and the fact that the polymer has been already used to successfully “wire” different redox enzymes in biosensors and biofuel cells [14, 46, 47], it was selected for this study. The possibility to successfully “wire” the other FADGDHs to graphite rod electrodes was therefore anticipated [43]. The mechanism of interaction and electron transfer between enzymes and Os-polymers is well described in the following reports [45, 48–50]. The FADGDHs belong to the same structural family of GMC oxidoreductases (glucose-methanol-choline oxidoreductase) as GOx [51]. Similar to GOx, the FADGDHs oxidize glucose at the C1 position and only the β-form is oxidized and 2e−/2H+ are transferred from glucose to the bound FAD, Reaction 1. The electron transfer occurs between FADGDHs and Os-polymers through the following reactions.Fig. 1

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