High current density "wired" quinoprotein glucose dehydrogenase electrode

Ye, Ling.; Haemmerle, Martin.; Olsthoorn, Arjen J. J.; Schuhmann, Wolfgang; Schmidt, H.‐L.; Duine, Johannis A.; Heller, Adam

doi:10.1021/ac00051a008

Cited by 179 publications

(61 citation statements)

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“…sGDH has so far been found only in A. calcoaceticus strains [5]. Although the enzyme is an extremely efficient catalyst for in vitro glucose oxidation, making it an attractive candidate for analytical applications [6], such a role is absent in whole cells, implying that the physiological function of sGDH is unknown [7]. Except for a small region, the amino acid sequence of sGDH [XI is completely different from mGDH and from those of the quinoprotein alcohol dehydrogenases [9].…”

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confidence: 99%

Ca²⁺ and its Substitutes have Two Different Binding Sites and Roles in Soluble, Quinoprotein (Pyrroloquinoline‐Quinone‐Containing) Glucose Dehydrogenase

Olsthoorn

Otsuki

Duine

1997

European Journal of Biochemistry

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To investigate the mode of binding and the role of Ca2+ in soluble, pyrroloquinoline-quinone (PQQ)-containing glucose dehydrogenase of the bacterium Acinetobacter calcoaceticus (sGDH), the following enzyme species were prepared and their interconversions studied : monomeric apoenzyme (M) ; monomer with one firmly bound Ca2+ ion (M*); dimer consisting of 2 M* (D); dimer consisting of 2 M and 2 PQQ (Holo-Y); dimer consisting of D with 2 PQQ (Holo-X); fully reconstituted enzyme consisting of Holo-X with two extra Ca2+ ions (Holo) or substitutes for Ca2+ (hybrid Holo-enzymes). D and Holo are very stable enzyme species regarding monomerization and inactivation by chelator, respectively, the bound CaZi being locked up in such a way that it is not accessible to chelator. D can be converted into M* by heat treatment and the tightly bound Ca'+ can be removed from M* with chelator, transforming it into M. Reassociation of M* to D occurs spontaneously at 20°C; reassociation of M to D occurs by adding a stoichiometric amount of Ca2+. Synergistic effects were exerted by bound Ca2+ and PQQ, each increasing the affinity of the protein for the other component. Dimerization of M to D occurred with Ca2', Cd2+, Mn2+, and Sr2+ (in decreasing order of effectiveness), but not with Mg", Ba2+, Cozi, Ni", Znz+, or monovalent cations. Conversion of inactive Holo-X into active Holo, was achieved with Ca2+ or metal ions effective in dimerization. Although it is likely that activation of Holo-X involves binding of metal ion to PQQ, the spectral and enzymatic activity differences between normal Holo-and hybrid Holo-enzymes are relatively small. Titration experiments revealed that the two Caz+ ions required for activation of Holo-X are even more firmly bound than the two required for dimerization of M and anchoring of PQQ. Although the two binding sites related with the dual function of Ca2+ show similar metal ion specificity, they are not identical. The presence of two different sites in sGDH appears to be unique because in other PQQ-containing dehydrogenases, the PQQ-containing subunit has only one site. Given the broad spectrum of bivalent metal ions effective in reconstituting quinoprotein dehydrogenase apoenzymes to active holoenzymes, but the limited spectrum for an individual enzyme, the specificity is not so much determined by PQQ but by the variable metal-ion-binding sites.Keywords: quinoprotein ; pyrroloquinoline quinone ; glucose dehydrogenase; reconstitution; calcium.The bacterium Acinetobacter calcoaceticus produces two quite different PQQ-containing (quinoprotein) glucose dehydrogenases, the membrane-bound enzyme (mGDH) and the soluble enzyme (sGDH). mGDH occurs in many gram-negative bacteria [l] and glucose oxidation via this enzyme provides the organism with useful energy [2]. The deduced amino acid sequence of this Correspondence to J. A. Duine,

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Ca²⁺ and its Substitutes have Two Different Binding Sites and Roles in Soluble, Quinoprotein (Pyrroloquinoline‐Quinone‐Containing) Glucose Dehydrogenase

Olsthoorn

Otsuki

Duine

1997

European Journal of Biochemistry

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“…This PQQ dependent has high catalytic activity (3) , however, the stability of the enzyme was somewhat inferior to GOD. It is necessary to obtain the thermostable enzyme.…”

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Amperometric Glucose Sensor Using Thermostable Co-Factor Binding Glucose Dehydrogenase

et al. 2003

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A thermostable mediator-type enzyme glucose sensor was constructed. The electrode was fabricated using chemically crosslinked thermostable co-factor binding glucose dehydrogenase (GDH) from thermophilic bacteria in carbon paste matrix. The electrode responded directly proportional to D-glucose concentration from 0.01 mM to 3 mM in stirred buffer containing 1 mM 1-methoxyphenazinemethosulfate as a mediator with the steady-state mode. The storage stability was examined by incubating the enzyme electrode at 50ºC during the measurement. The cross-linked GDH immobilized electrode showed good storage stability. Ninety percent of its initial response was retained after incubation in buffer solution for 9 days at 50ºC. The flow injection analysis (FIA) glucose sensing system was also constructed by immobilizing the cross-linked GDH and ferrocene as a mediator in the carbon paste matrix. The FIA system was able to measure 600 samples for 100 h.

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“…In order to elucidate the region responsible for PQQ binding, we have focused on one set of PQQGDH structural genes; E. coli PQQGDH, which easily loses its prosthetic group in the presence of EDTA, and A. calocoaceticus PQQGDH, which is very stable during EDTA treatment. Based on homology analysis of the putative PQQ binding site, we previously reported a mutant constructed by site directed mutagenesis with increased EDTA tolerance [6]. We have also succeeded in the construction of various chimeric PQQGDHs which differ in EDTA tolerance, by homologous recombination of E. coli and A. calcoaceticus PQQGDH structural genes ( [8] and Sode et al, submitted for *Corresponding author.…”

Section: Introductionmentioning

confidence: 99%

Thermostable chimeric PQQ glucose dehydrogenase

et al. 1995

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The thermal stability of PQQ glucose dehydrogenases (PQQGDHs) which were chimeras with more than 95% made up of the N-terminal region of Escherichia coli PQQGDH and the rest made up of the C-terminal region of Acinetobacter calcoaceticus PQQGDH was investigated. Among the chimeric PQQGDHs, E97A3 (E. eoli 97% and A. calcoaceticus 3%) and E95A5 were found to possess higher thermal stability than parental E. coli PQQGDH. Further detailed characterization of the thermal stability was carried out, focusing on E97A3. E97A3 showed a more than 3-fold and 12-fold increase in half life time at 40°C, compared with the PQQGDHs of E. coli and A. calcoaceticus, respectively. Using transition state theory, the increase in the free energy of inactivation observed in E97A3 was compared with those of the E. coli and ,4. calcoaceticus parental enzymes. The region responsible for this stabilization was also discussed.

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High current density "wired" quinoprotein glucose dehydrogenase electrode

Cited by 179 publications

References 16 publications

Ca²⁺ and its Substitutes have Two Different Binding Sites and Roles in Soluble, Quinoprotein (Pyrroloquinoline‐Quinone‐Containing) Glucose Dehydrogenase

Ca²⁺ and its Substitutes have Two Different Binding Sites and Roles in Soluble, Quinoprotein (Pyrroloquinoline‐Quinone‐Containing) Glucose Dehydrogenase

Amperometric Glucose Sensor Using Thermostable Co-Factor Binding Glucose Dehydrogenase

Thermostable chimeric PQQ glucose dehydrogenase

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High current density "wired" quinoprotein glucose dehydrogenase electrode

Cited by 179 publications

References 16 publications

Ca2+ and its Substitutes have Two Different Binding Sites and Roles in Soluble, Quinoprotein (Pyrroloquinoline‐Quinone‐Containing) Glucose Dehydrogenase

Ca2+ and its Substitutes have Two Different Binding Sites and Roles in Soluble, Quinoprotein (Pyrroloquinoline‐Quinone‐Containing) Glucose Dehydrogenase

Amperometric Glucose Sensor Using Thermostable Co-Factor Binding Glucose Dehydrogenase

Thermostable chimeric PQQ glucose dehydrogenase

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Product

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Ca²⁺ and its Substitutes have Two Different Binding Sites and Roles in Soluble, Quinoprotein (Pyrroloquinoline‐Quinone‐Containing) Glucose Dehydrogenase

Ca²⁺ and its Substitutes have Two Different Binding Sites and Roles in Soluble, Quinoprotein (Pyrroloquinoline‐Quinone‐Containing) Glucose Dehydrogenase