Electron-Transfer Studies with a New Flavin Adenine Dinucleotide Dependent Glucose Dehydrogenase and Osmium Polymers of Different Redox Potentials

A new extracellular flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase from Glomerella cingulata (<i>Gc</i>GDH) 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” <i>Gc</i>GDH to the spectrographic graphite electrode’s surface. The <i>Gc</i>GDH/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 <i>Gc</i>GDH/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)Cl]⁺ had a potential of +309 mV versus NHE, and the optimum <i>Gc</i>GDH/Os polymer ratio was 1:2 yielding a maximum current density of 493 μA·cm^–2 at a 30 mM glucose concentration

Purification of recombinant GalOx expressed in <i>E. coli.</i>

<p>Purification of recombinant GalOx expressed in <i>E. coli.</i></p

Alignment of GalOx from <i>F. oxysporum</i> and <i>F. graminearum</i>.

<p>The prepro sequence is underlined. Amino acid residues involved in copper binding are highlighted.</p

3D Structure of GalOx of <i>F. oxysporum</i>.

<p>A: Overall structure showing the predominantly β-structure. The N-terminus, C-terminus and the copper atom in the active site are highlighted. B: The active site of GalOx showing the copper ligands and the thioether cross-link. The structural model was generated by homology modelling based on the published structure of mature GalOx from <i>F. graminearum</i> (PDB 1gog) using SWISS_MODEL.</p

Apparent kinetic constants of GalOx produced in <i>E. coli</i> for several electron donor substrates.

<p>Apparent kinetic constants of GalOx produced in <i>E. coli</i> for several electron donor substrates.</p

MALDI-TOF peptide mass map of GalOx expressed in <i>E. coli</i>.

<p>The peptides were generated by sequential digestion using trypsin and Asp-N. The peak labels correspond to the [M+H]⁺ ions of the obtained peptide fragments and their positions in the GalOx sequence. The spectrum also shows two intense signals (marked with asterisk) at m/z 2237.9 and 2374.9 related to the cross-linked peptides 266–274/312–323 and 265–274/312–323, respectively. The identity of the cross-linked peptides was verified by MS/MS fragmentation.</p

MALDI-FTMS data of the cross-linked peptides produced by in-gel digestion of GalOx. The cross-linked amino acids C230 and Y274 are highlighted.

<p>MALDI-FTMS data of the cross-linked peptides produced by in-gel digestion of GalOx. The cross-linked amino acids C230 and Y274 are highlighted.</p

Active clones (in %) of site-saturation mutagenesis target positions.

<p>Activity was determined using a screening assay containing 0.3-glucose in 100 mM sodium acetate buffer pH 4 at 30°C. Libraries were expressed in <i>S. cerevisiae</i>. wt: wild-type <i>A. meleagris</i> PDH.</p

Temperature dependence of the activity of GalOx expressed in <i>E. coli.</i>

<p>Temperature dependence of the activity of GalOx expressed in <i>E. coli.</i></p

Effect of the pH on the activity of GalOx expressed in <i>E. coli</i>.

<p>The buffers used were 50(♦), 50 mM phosphate (_▀) and 50 mM Tris (▴).</p