The Experts below are selected from a list of 327 Experts worldwide ranked by ideXlab platform
Seiya Tsujimura - One of the best experts on this subject based on the ideXlab platform.
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toward an ideal platform structure based on mgo templated carbon for flavin adenine dinucleotide dependent Glucose Dehydrogenase os polymer hydrogel electrodes
Electrochimica Acta, 2020Co-Authors: Seiya Tsujimura, Satoshi TakeuchiAbstract:Abstract For the efficient electrochemical Glucose oxidation on the electrode modified with redox hydrogel involving Os-tethered polymer and flavin adenine dinucleotide-dependent Glucose Dehydrogenase (FADGDH), the effect of pore size of MgO-templated carbon (MgOC) as an electrode material was studied. The MgOC was modified on glassy carbon electrode by ink-drop-casting technique. The MgOC pore size clearly affected on the current generation efficiency for Glucose oxidation. As the pore size increased above 100 nm, a Glucose oxidation current density of more than 100 mA cm−2 was achieved with 1000 μg cm−2 of hydrogel loading. Both high specific surface area and macrostructure of MgOC, which does not impede mass transport even if hydrogel loading is increased, are important factors in designing the porous structure of the MgOC layer via the ink-drop-casting process during electrode fabrication.
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mediated electrochemical oxidation of Glucose via poly methylene green grafted on the carbon surface catalyzed by flavin adenine dinucleotide dependent Glucose Dehydrogenase
Colloids and Surfaces B: Biointerfaces, 2020Co-Authors: Nozomu Tsuruoka, Silvia Sato Soto, Awatef Ben Tahar, Abdelkader Zebda, Seiya TsujimuraAbstract:Abstract Electrochemically polymerized phenothiazines (thionine, methylene green, methylene blue, and toluidine blue) on carbon electrodes were investigated as electron transfer mediators of Glucose oxidation by flavin adenine dinucleotide-dependent Glucose Dehydrogenase (FAD-GDH) for biosensor and biofuel cell applications. Among the tested polyphenothiazines grafted on a glassy carbon electrode, clear redox-mediating activity was observed for poly(methylene green), and the catalytic oxidation current depended on the concentrations of Glucose and enzymes and the amount of polymer deposited on the electrode surface. The poly(methylene green)-grafted porous carbon electrodes showed 3 mA cm−2 of Glucose oxidation current catalyzed by FAD-GDH.
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exceptionally high Glucose current on a hierarchically structured porous carbon electrode with wired flavin adenine dinucleotide dependent Glucose Dehydrogenase
Journal of the American Chemical Society, 2014Co-Authors: Seiya Tsujimura, Kazuki Murata, Wataru AkatsukaAbstract:This article introduces a carbon electrode designed to achieve efficient enzymatic electrolysis by exploiting a hierarchical pore structure based on macropores for efficient mass transfer and mesopores for high enzyme loading. Magnesium oxide-templated mesoporous carbon (MgOC, mean pore diameter 38 nm) was used to increase the effective specific surface area for enzyme immobilization. MgOC particles were deposited on a current collector by an electrophoretic deposition method to generate micrometer-scale macropores to improve the mass transfer of Glucose and electrolyte (buffer) ions. To create a Glucose bioanode, the porous-carbon-modified electrode was further coated with a biocatalytic hydrogel composed of a conductive redox polymer, deglycosylated flavin adenine dinucleotide-dependent Glucose Dehydrogenase (d-FAD-GDH), and a cross-linker. Carbohydrate chains on the peripheral surfaces of the FAD-GDH molecules were removed by periodate oxidation before cross-linking. The current density for the oxidati...
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Glucose oxidation catalyzed by fad dependent Glucose Dehydrogenase within os complex tethered redox polymer hydrogel
Electrochimica Acta, 2014Co-Authors: Kazuki Murata, Wataru Akatsuka, Takuya Sadakane, Aya Matsunaga, Seiya TsujimuraAbstract:Abstract FAD-dependent Glucose Dehydrogenase (FAD-GDH) from Aspergillus terreus was co-immobilized on a glassy carbon (GC) electrode surface with a poly(1-vinylimidazole)-tethered Os(2,2′-bipyridine) 2 Cl complex as a redox mediator. The steady-state catalytic current for Glucose oxidation was 2.6 mA cm −2 at pH 7 and 25 °C. This value increased 1.6-fold after oxidative deglycosylation of the enzyme, which is the highest value so far reported for a GC-electrode-based Glucose anode. The deglycosylation process did not decrease the stability of the FAD-GDH dissolved in the buffer solution and immobilized within the hydrogel. A 10% decrease in the catalytic current was observed after 24 h continuous operation.
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novel fad dependent Glucose Dehydrogenase for a dioxygen insensitive Glucose biosensor
Bioscience Biotechnology and Biochemistry, 2006Co-Authors: Seiya Tsujimura, Shinki Kojima, Kenji Kano, Tokuji Ikeda, Mika Sato, Hirokazu Sanada, Hironori OmuraAbstract:A novel FAD-dependent Glucose Dehydrogenase (FAD-GDH) was found and its enzymatic property for Glucose sensing was characterized. FAD-GDH oxidized Glucose in the presence of some artificial electron acceptors, except for O2, and exhibited thermostability, high substrate specificity and a large Michaelis constant for Glucose. FAD-GDH was applied to an amperometric Glucose sensor with Fe(CN)6(3-) as a soluble mediator. The use of a relatively high concentration of Fe(CN)6(3-) resulted in a good linearity between the current response and the Glucose concentration, taking into account a large Michaelis constant for Fe(CN)6(3-). The Glucose sensor was completely insensitive to O2 and responded linearly to Glucose up to 30 mM. Compared to Glucose, the response to other saccharides was negligible. The sensor can be stored at room temperature in a desiccator for at least one month without any change in the response or activity.
Koji Sode - One of the best experts on this subject based on the ideXlab platform.
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x ray structure of the direct electron transfer type fad Glucose Dehydrogenase catalytic subunit complexed with a hitchhiker protein
Acta crystallographica. Section D Structural biology, 2019Co-Authors: Hiromi Yoshida, Koji Sode, Stefano Ferri, Wakako Tsugawa, Katsuhiro Kojima, Masaki Shiota, Keiichi Yoshimatsu, Tomohiko Yamazaki, Shigehiro KamitoriAbstract:The bacterial flavin adenine dinucleotide (FAD)-dependent Glucose Dehydrogenase complex derived from Burkholderia cepacia (BcGDH) is a representative molecule of direct electron transfer-type FAD-dependent Dehydrogenase complexes. In this study, the X-ray structure of BcGDHγα, the catalytic subunit (α-subunit) of BcGDH complexed with a hitchhiker protein (γ-subunit), was determined. The most prominent feature of this enzyme is the presence of the 3Fe–4S cluster, which is located at the surface of the catalytic subunit and functions in intramolecular and intermolecular electron transfer from FAD to the electron-transfer subunit. The structure of the complex revealed that these two molecules are connected through disulfide bonds and hydrophobic interactions, and that the formation of disulfide bonds is required to stabilize the catalytic subunit. The structure of the complex revealed the putative position of the electron-transfer subunit. A comparison of the structures of BcGDHγα and membrane-bound fumarate reductases suggested that the whole BcGDH complex, which also includes the membrane-bound β-subunit containing three heme c moieties, may form a similar overall structure to fumarate reductases, thus accomplishing effective electron transfer.
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novel fungal fad Glucose Dehydrogenase derived from aspergillus niger for Glucose enzyme sensor strips
Biosensors and Bioelectronics, 2017Co-Authors: Koji Sode, Wakako Tsugawa, Noya Loew, Yosuke Ohnishi, Hayato Tsuruta, Kazushige Mori, Katsuhiro Kojima, Jeffrey T Labelle, David C KlonoffAbstract:In this study, a novel fungus FAD dependent Glucose Dehydrogenase, derived from Aspergillus niger (AnGDH), was characterized. This enzyme's potential for the use as the enzyme for blood Glucose monitor enzyme sensor strips was evaluated, especially by investigating the effect of the presence of xylose during Glucose measurements. The substrate specificity of AnGDH towards Glucose was investigated, and only xylose was found as a competing substrate. The specific catalytic efficiency for xylose compared to Glucose was 1.8%. The specific activity of AnGDH for xylose at 5mM concentration compared to Glucose was 3.5%. No other sugars were used as substrate by this enzyme. The superior substrate specificity of AnGDH was also demonstrated in the performance of enzyme sensor strips. The impact of spiking xylose in a sample with physiological Glucose concentrations on the sensor signals was investigated, and it was found that enzyme sensor strips using AnGDH were not affected at all by 5mM (75mg/dL) xylose. This is the first report of an enzyme sensor strip using a fungus derived FADGDH, which did not show any positive bias at a therapeutic level xylose concentration on the signal for a Glucose sample. This clearly indicates the superiority of AnGDH over other conventionally used fungi derived FADGDHs in the application for SMBG sensor strips. The negligible activity of AnGDH towards xylose was also explained on the basis of a 3D structural model, which was compared to the 3D structures of A. flavus derived FADGDH and of two Glucose oxidases.
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site directed mutagenesis studies of fad dependent Glucose Dehydrogenase catalytic subunit of burkholderia cepacia
Biotechnology Letters, 2008Co-Authors: Hideaki Yamaoka, Yuki Yamashita, Stefano Ferri, Koji SodeAbstract:A FAD-dependent Glucose Dehydrogenase (FADGDH) mutant with narrow substrate specificity was constructed by site-directed mutagenesis. Several characteristics of FADGDH, such as high catalytic activity and high electron transfer ability, make this enzyme suitable for application to Glucose sensors. However, for further applications, improvement of the broad substrate specificity is needed. In this paper, we mutated two residues, Asn475 and Ala472, which are located near the putative active site of the catalytic subunit of FADGDH and have been predicted from the alignment with the active site of Glucose oxidase. Of the 38 mutants constructed, Ala472Phe and Asn475Asp were purified and their activities were analyzed. Both mutants showed a higher specificity toward Glucose compared to the wild type enzyme.
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cloning and functional expression of Glucose Dehydrogenase complex of burkholderia cepacia in escherichia coli
Journal of Biotechnology, 2006Co-Authors: Taiki Tsuya, Stefano Ferri, Hideaki Yamaoka, Masako Fujikawa, Koji SodeAbstract:The thermostable Glucose Dehydrogenase (GDH) from Burkholderia cepacia sp. SM4 is composed of a catalytic subunit (alpha), an electron transfer subunit (beta), and a small gamma subunit of unknown function. We cloned a 1428-nucleotide gene encoding the beta subunit located immediately downstream of the alpha subunit. This completes the isolation of the genes encoding the three components of the GDH complex, which are clustered very close together with the same transcription polarity in the order gammaalphabeta. The deduced beta subunit amino acid sequence contains three typical heme-binding motifs and was 44-49% identical to the cytochrome c subunits of other FAD-dependent Dehydrogenase complexes. The GDHgammaalphabeta complex of B. cepacia was successfully expressed in a fully active form in Escherichia coli by co-expression with cytochrome c maturation genes. Recombinant expression of the GDH complex was also found to restore Glucose-dependent respiration in a GDH mutant of E. coli.
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development of a novel Glucose enzyme fuel cell system employing protein engineered pqq Glucose Dehydrogenase
Biosensors and Bioelectronics, 2005Co-Authors: Noriko Yuhashi, Junko Okuda, Satoshi Igarashi, Masamitsu Tomiyama, Kazunori Ikebukuro, Koji SodeAbstract:Abstract Glucose Dehydrogenase harboring pyrroloquinoline quinone as the prosthetic group (PQQGDH) from Acinetobacter calcoaceticus is an ideal enzyme for the anode of biofuel cell, because of its oxygen insensitivity and high catalytic efficiency. However, the application of PQQGDH for the bioanode is inherently limited because of its instability. Using Ser415Cys mutant whose stability was greatly improved, we constructed the biofuel cell system employing the engineered PQQGDH as the bioanode enzyme and bilirubin oxidase (BOD) as the biocathode, and compared the stability of the biofuel cell with that employing wild-type PQQGDH. The maximum power density was 17.6 μW/cm 2 at an external optimal load of 200 kΩ. Using Ser415Cys mutant, the lifetime of the biofuel cell system was greatly extended to 152 h, more than six times as that of the biofuel cell employing the wild-type.
María José Bonete - One of the best experts on this subject based on the ideXlab platform.
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alteration of coenzyme specificity in halophilic nad p Glucose Dehydrogenase by site directed mutagenesis
Journal of Molecular Catalysis B-enzymatic, 2009Co-Authors: Carmen Pire, Juan Ferrer, J. Esclapez, Susana Diaz, Francisco Perezpomares, María José BoneteAbstract:Abstract Structural analysis of Glucose Dehydrogenase from Haloferax mediterranei revealed that the adenosine 2′-phosphate of NADP + was stabilized by the side chains of Arg207 and Arg208. To investigate the structural determinants for coenzyme specificity, several mutants involving residues Gly206, Arg207 and Arg208 were engineered and kinetically characterized. The single mutants G206D and R207I were less efficient with NADP + than the wild type, and the double and triple mutants G206D/R207I and G206D/R207I/R208N showed no activity with NADP + . In the single mutant G206D, the relation k cat / K NA D + was 1.6 times higher than in the wild type, resulting in an enzyme that preferred NAD + over NADP + . The single mutation was sufficient to modify coenzyme specificity, whereas other Dehydrogenases usually required more than one or two mutations to change coenzyme specificity. However, the highest reaction rates were reached with the double mutant G206D/R207I and with coenzyme NAD + , where the k cat was 1.6 times higher than the k cat of the wild-type enzyme with NADP + . However, catalytic efficiency with NAD + was lower, as the K m value for coenzyme was 77 times higher than the wild type with NADP + .
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analysis of acidic surface of haloferax mediterranei Glucose Dehydrogenase by site directed mutagenesis
FEBS Letters, 2007Co-Authors: J. Esclapez, Carmen Pire, Juan Ferrer, Rosa Maria Martinezespinosa, Vanesa Bautista, María José BoneteAbstract:Generally, halophilic enzymes present a characteristic amino acid composition, showing an increase in the content of acidic residues and a decrease in the content of basic residues, particularly lysines. The latter decrease appears to be responsible for a reduction in the proportion of solvent-exposed hydrophobic surface. This role was investigated by site-directed mutagenesis of Glucose Dehydrogenase from Haloferax mediterranei, in which surface aspartic residues were changed to lysine residues. From the biochemical analysis of the mutant proteins, it is concluded that the replacement of the aspartic residues by lysines results in slightly less halotolerant proteins, although they retain the same enzymatic activities and kinetic parameters compared to the wild type enzyme.
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Analysis of protein solvent interactions in Glucose Dehydrogenase from the extreme halophile Haloferax mediterranei
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: K. Linda Britton, Carmen Pire, María José Bonete, Juan Ferrer, J. Esclapez, Patrick J. Baker, M. Fisher, Sergey N. Ruzheinikov, D. James Gilmour, David W. RiceAbstract:The structure of Glucose Dehydrogenase from the extreme halophile Haloferax mediterranei has been solved at 1.6-A resolution under crystallization conditions which closely mimic the "in vivo" intracellular environment. The decoration of the enzyme's surface with acidic residues is only partially neutralized by bound potassium counterions, which also appear to play a role in substrate binding. The surface shows the expected reduction in hydrophobic character, surprisingly not from changes associated with the loss of exposed hydrophobic residues but rather arising from a loss of lysines consistent with the genome wide-reduction of this residue in extreme halophiles. The structure reveals a highly ordered, multilayered solvation shell that can be seen to be organized into one dominant network covering much of the exposed surface accessible area to an extent not seen in almost any other protein structure solved. This finding is consistent with the requirement of the enzyme to form a protective shell in a dehydrating environment.
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crystallization and preliminary x ray analysis of binary and ternary complexes of haloferax mediterranei Glucose Dehydrogenase
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2005Co-Authors: J. Esclapez, Carmen Pire, María José Bonete, Juan Ferrer, Patrick J. Baker, M. Fisher, K L Britton, David W. RiceAbstract:Haloferax mediterranei Glucose Dehydrogenase (EC 1.1.1.47) belongs to the medium-chain alcohol Dehydrogenase superfamily and requires zinc for catalysis. In the majority of these family members, the catalytic zinc is tetrahedrally coordinated by the side chains of a cysteine, a histidine, a cysteine or glutamate and a water molecule. In H. mediterranei Glucose Dehydrogenase, sequence analysis indicates that the zinc coordination is different, with the invariant cysteine replaced by an aspartate residue. In order to analyse the significance of this replacement and to contribute to an understanding of the role of the metal ion in catalysis, a range of binary and ternary complexes of the wild-type and a D38C mutant protein have been crystallized. For most of the complexes, crystals belonging to space group I222 were obtained using sodium/potassium citrate as a precipitant. However, for the binary and non-productive ternary complexes with NADPH/Zn, it was necessary to replace the citrate with 2-methyl-2,4-pentanediol. Despite the radical change in conditions, the crystals thus formed were isomorphous.
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stability and enzymatic studies of Glucose Dehydrogenase from the archaeon haloferax mediterranei in reverse micelles
Biocatalysis and Biotransformation, 2004Co-Authors: Carmen Pire, Juan Ferrer, J. Esclapez, Frutos C Marhuendaegea, Luis A Alcaraz, María José BoneteAbstract:Reverse micelles were used as a cytoplasmic model to study the kinetics of an extreme halophilic enzyme such as the recombinant Glucose Dehydrogenase from the Archaeon Haloferax mediterranei. This enzyme was solubilized in reverse micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as co-surfactant. Glucose Dehydrogenase retained its catalytic properties in this organic medium, showing good stability at low water content, even at low salt concentration (125 mM NaCl). The dependence of the enzymatic activity on the molar water surfactant ratio (w0=[H2O]/[surfactant]) increased with rising water content. Surprisingly, the activity of this extreme halophilic enzyme did not depend on the salt concentration in reverse micelles. The kinetic of the enzymatic oxidation of β-D-Glucose to D-glucono-1,5-lactone using NADP+ as coenzyme for the Glucose Dehydrogenase from Haloferax mediterranei was also studied in the reverse micellar system.
Carmen Pire - One of the best experts on this subject based on the ideXlab platform.
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alteration of coenzyme specificity in halophilic nad p Glucose Dehydrogenase by site directed mutagenesis
Journal of Molecular Catalysis B-enzymatic, 2009Co-Authors: Carmen Pire, Juan Ferrer, J. Esclapez, Susana Diaz, Francisco Perezpomares, María José BoneteAbstract:Abstract Structural analysis of Glucose Dehydrogenase from Haloferax mediterranei revealed that the adenosine 2′-phosphate of NADP + was stabilized by the side chains of Arg207 and Arg208. To investigate the structural determinants for coenzyme specificity, several mutants involving residues Gly206, Arg207 and Arg208 were engineered and kinetically characterized. The single mutants G206D and R207I were less efficient with NADP + than the wild type, and the double and triple mutants G206D/R207I and G206D/R207I/R208N showed no activity with NADP + . In the single mutant G206D, the relation k cat / K NA D + was 1.6 times higher than in the wild type, resulting in an enzyme that preferred NAD + over NADP + . The single mutation was sufficient to modify coenzyme specificity, whereas other Dehydrogenases usually required more than one or two mutations to change coenzyme specificity. However, the highest reaction rates were reached with the double mutant G206D/R207I and with coenzyme NAD + , where the k cat was 1.6 times higher than the k cat of the wild-type enzyme with NADP + . However, catalytic efficiency with NAD + was lower, as the K m value for coenzyme was 77 times higher than the wild type with NADP + .
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analysis of acidic surface of haloferax mediterranei Glucose Dehydrogenase by site directed mutagenesis
FEBS Letters, 2007Co-Authors: J. Esclapez, Carmen Pire, Juan Ferrer, Rosa Maria Martinezespinosa, Vanesa Bautista, María José BoneteAbstract:Generally, halophilic enzymes present a characteristic amino acid composition, showing an increase in the content of acidic residues and a decrease in the content of basic residues, particularly lysines. The latter decrease appears to be responsible for a reduction in the proportion of solvent-exposed hydrophobic surface. This role was investigated by site-directed mutagenesis of Glucose Dehydrogenase from Haloferax mediterranei, in which surface aspartic residues were changed to lysine residues. From the biochemical analysis of the mutant proteins, it is concluded that the replacement of the aspartic residues by lysines results in slightly less halotolerant proteins, although they retain the same enzymatic activities and kinetic parameters compared to the wild type enzyme.
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Analysis of protein solvent interactions in Glucose Dehydrogenase from the extreme halophile Haloferax mediterranei
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: K. Linda Britton, Carmen Pire, María José Bonete, Juan Ferrer, J. Esclapez, Patrick J. Baker, M. Fisher, Sergey N. Ruzheinikov, D. James Gilmour, David W. RiceAbstract:The structure of Glucose Dehydrogenase from the extreme halophile Haloferax mediterranei has been solved at 1.6-A resolution under crystallization conditions which closely mimic the "in vivo" intracellular environment. The decoration of the enzyme's surface with acidic residues is only partially neutralized by bound potassium counterions, which also appear to play a role in substrate binding. The surface shows the expected reduction in hydrophobic character, surprisingly not from changes associated with the loss of exposed hydrophobic residues but rather arising from a loss of lysines consistent with the genome wide-reduction of this residue in extreme halophiles. The structure reveals a highly ordered, multilayered solvation shell that can be seen to be organized into one dominant network covering much of the exposed surface accessible area to an extent not seen in almost any other protein structure solved. This finding is consistent with the requirement of the enzyme to form a protective shell in a dehydrating environment.
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crystallization and preliminary x ray analysis of binary and ternary complexes of haloferax mediterranei Glucose Dehydrogenase
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2005Co-Authors: J. Esclapez, Carmen Pire, María José Bonete, Juan Ferrer, Patrick J. Baker, M. Fisher, K L Britton, David W. RiceAbstract:Haloferax mediterranei Glucose Dehydrogenase (EC 1.1.1.47) belongs to the medium-chain alcohol Dehydrogenase superfamily and requires zinc for catalysis. In the majority of these family members, the catalytic zinc is tetrahedrally coordinated by the side chains of a cysteine, a histidine, a cysteine or glutamate and a water molecule. In H. mediterranei Glucose Dehydrogenase, sequence analysis indicates that the zinc coordination is different, with the invariant cysteine replaced by an aspartate residue. In order to analyse the significance of this replacement and to contribute to an understanding of the role of the metal ion in catalysis, a range of binary and ternary complexes of the wild-type and a D38C mutant protein have been crystallized. For most of the complexes, crystals belonging to space group I222 were obtained using sodium/potassium citrate as a precipitant. However, for the binary and non-productive ternary complexes with NADPH/Zn, it was necessary to replace the citrate with 2-methyl-2,4-pentanediol. Despite the radical change in conditions, the crystals thus formed were isomorphous.
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stability and enzymatic studies of Glucose Dehydrogenase from the archaeon haloferax mediterranei in reverse micelles
Biocatalysis and Biotransformation, 2004Co-Authors: Carmen Pire, Juan Ferrer, J. Esclapez, Frutos C Marhuendaegea, Luis A Alcaraz, María José BoneteAbstract:Reverse micelles were used as a cytoplasmic model to study the kinetics of an extreme halophilic enzyme such as the recombinant Glucose Dehydrogenase from the Archaeon Haloferax mediterranei. This enzyme was solubilized in reverse micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as co-surfactant. Glucose Dehydrogenase retained its catalytic properties in this organic medium, showing good stability at low water content, even at low salt concentration (125 mM NaCl). The dependence of the enzymatic activity on the molar water surfactant ratio (w0=[H2O]/[surfactant]) increased with rising water content. Surprisingly, the activity of this extreme halophilic enzyme did not depend on the salt concentration in reverse micelles. The kinetic of the enzymatic oxidation of β-D-Glucose to D-glucono-1,5-lactone using NADP+ as coenzyme for the Glucose Dehydrogenase from Haloferax mediterranei was also studied in the reverse micellar system.
Roland Ludwig - One of the best experts on this subject based on the ideXlab platform.
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electron transfer studies with a new flavin adenine dinucleotide dependent Glucose Dehydrogenase and osmium polymers of different redox potentials
Analytical Chemistry, 2012Co-Authors: Muhammad Nadeem Zafar, Christoph Sygmund, Roland Ludwig, Xiaoju Wang, Donal Leech, Lo GortonAbstract:A new extracellular flavin adenine dinucleotide (FAD)-dependent Glucose Dehydrogenase from Glomerella cingulata (GcGDH) was electrochemically studied as a recognition element in Glucose biosensors. The redox enzyme was recombinantly produced in Pichia pastoris and homogeneously purified, and its Glucose-oxidizing properties on spectrographic graphite electrodes were investigated. Six different Os polymers, the redox potentials of which ranged in a broad potential window between +15 and +489 mV versus the normal hydrogen electrode (NHE), were used to immobilize and "wire" GcGDH to the spectrographic graphite electrode's surface. The GcGDH/Os polymer modified electrodes were evaluated by chronoamperometry using flow injection analysis. The current response was investigated using a stepwisely increased applied potential. It was observed that the ratio of GcGDH/Os polymer and the overall loading of the enzyme electrode significantly affect the performance of the enzyme electrode for Glucose oxidation. The best-suited Os polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)CI](+) had a potential of +309 mV versus NHE, and the optimum GcGDH/Os polymer ratio was 1:2 yielding a maximum current density of 493,mu A.cm(-2) at a 30 mM Glucose concentration. (Less)
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heterologous overexpression of glomerella cingulata fad dependent Glucose Dehydrogenase in escherichia coli and pichia pastoris
Microbial Cell Factories, 2011Co-Authors: Christoph Sygmund, Petra Staudigl, Miriam Klausberger, Nikos Pinotsis, Kristina Djinoviccarugo, Dietmar Haltrich, Roland LudwigAbstract:Background FAD dependent Glucose Dehydrogenase (GDH) currently raises enormous interest in the field of Glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into Glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by Glomerella (anamorph Colletotrichum) spp.
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heterologous overexpression of glomerella cingulata fad dependent Glucose Dehydrogenase in escherichia coli and pichia pastoris
Microbial Cell Factories, 2011Co-Authors: Christoph Sygmund, Petra Staudigl, Miriam Klausberger, Nikos Pinotsis, Kristina Djinoviccarugo, Dietmar Haltrich, Lo Gorton, Roland LudwigAbstract:FAD dependent Glucose Dehydrogenase (GDH) currently raises enormous interest in the field of Glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into Glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by Glomerella (anamorph Colletotrichum) spp. Heterologous expression of active G. cingulata GDH has been achieved in both Escherichia coli and Pichia pastoris, however, the expressed volumetric activity was about 4800-fold higher in P. pastoris. Expression in E. coli resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a P. pastoris transformant resulted in an expression of 48,000 U L-1 of GDH activity (57 mg L-1). Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in P. pastoris and the wild-type enzyme from its natural fungal source. The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in P. pastoris facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies.
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reduction of quinones and phenoxy radicals by extracellular Glucose Dehydrogenase from glomerella cingulata suggests a role in plant pathogenicity
Microbiology, 2011Co-Authors: Christoph Sygmund, Miriam Klausberger, Alfons K G Felice, Roland LudwigAbstract:The plant-pathogenic fungus Glomerella cingulata (anamorph Colletotrichum gloeosporoides) secretes high levels of an FAD-dependent Glucose Dehydrogenase (GDH) when grown on tomato juice-supplemented media. To elucidate its molecular and catalytic properties, GDH was produced in submerged culture. The highest volumetric activity was obtained in shaking flasks after 6 days of cultivation (3400 U l−1, 4.2 % of total extracellular protein). GDH is a monomeric protein with an isoelectric point of 5.6. The molecular masses of the glycoforms ranged from 95 to 135 kDa, but after deglycosylation, a single 68 kDa band was obtained. The absorption spectrum is typical for an FAD-containing enzyme with maxima at 370 and 458 nm and the cofactor is non-covalently bound. The preferred substrates are Glucose and xylose. Suitable electron acceptors are quinones, phenoxy radicals, 2,6-dichloroindophenol, ferricyanide and ferrocenium hexafluorophosphate. In contrast, oxygen turnover is very low. The GDH-encoding gene was cloned and phylogenetic analysis of the translated protein reveals its affiliation to the GMC family of oxidoreductases. The proposed function of this quinone and phenoxy radical reducing enzyme is to neutralize the action of plant laccase, phenoloxidase or peroxidase activities, which are increased in infected plants to evade fungal attack.