Mercury pollution for marine environment at Farwa Island, Libya

Coimmobilization of pyranose dehydrogenase as an enzyme catalyst, osmium redox polymers [Os­(4,4′-dimethoxy-2,2′-bipyridine)₂(poly­(vinylimidazole))₁₀Cl]⁺ or [Os­(4,4′-dimethyl-2,2′-bipyridine)₂(poly­(vinylimidazole))₁₀Cl]⁺ as mediators, and carbon nanotube conductive scaffolds in films on graphite electrodes provides enzyme electrodes for glucose oxidation. The recombinant enzyme and a deglycosylated form, both expressed in Pichia pastoris, are investigated and compared as biocatalysts for glucose oxidation using flow injection amperometry and voltammetry. In the presence of 5 mM glucose in phosphate-buffered saline (PBS) (50 mM phosphate buffer solution, pH 7.4, with 150 mM NaCl), higher glucose oxidation current densities, 0.41 mA cm^–2, are obtained from enzyme electrodes containing the deglycosylated form of the enzyme. The optimized glucose-oxidizing anode, prepared using deglycosylated enzyme coimmobilized with [Os­(4,4′-dimethyl-2,2′-bipyridine)₂(poly­(vinylimidazole))₁₀Cl]⁺ and carbon nanotubes, was coupled with an oxygen-reducing bilirubin oxidase on gold nanoparticle dispersed on gold electrode as a biocathode to provide a membraneless fully enzymatic fuel cell. A maximum power density of 275 μW cm^–2 is obtained in 5 mM glucose in PBS, the highest to date under these conditions, providing sufficient power to enable wireless transmission of a signal to a data logger. When tested in whole human blood and unstimulated human saliva maximum power densities of 73 and 6 μW cm^–2 are obtained for the same fuel cell configuration, respectively

Characterization of nanoporous gold electrodes for bioelectrochemical applications

The high surface areas of nanostructured electrodes can provide for significantly enhanced surface loadings of electroactive materials. The fabrication and characterization of nanoporous gold (np-Au) substrates as electrodes for bioelectrochemical applications is described. Robust np-Au electrodes were prepared by sputtering a gold-silver alloy onto a glass support and subsequent dealloying of the silver component. Alloy layers were prepared with either a uniform or nonuniform distribution of silver and, post dealloying, showed clear differences in morphology on characterization with scanning electron microscopy. Redox reactions under kinetic control, in particular measurement of the charge required to strip a gold oxide layer, provided the most accurate measurements of the total electrochemically addressable electrode surface area, A(real). Values of A(real) up to 28 times that of the geometric electrode surface area, A(geo), were obtained. For diffusion-controlled reactions, overlapping diffusion zones between adjacent nanopores established limiting semi-infinite linear diffusion fields where the maximum current density was dependent on A(geo). The importance of measuring the surface area available for the immobilization was determined using the redox protein, cyt c. The area accessible to modification by a biological macromolecule, A(macro), such as cyt c was reduced by up to 40% compared to A(real), demonstrating that the confines of some nanopores were inaccessible to large macromolecules due to steric hindrances. Preliminary studies on the preparation of np-Au electrodes modified with osmium redox polymer hydrogels and Myrothecium verrucaria bilirubin oxidase (MvBOD) as a biocathode were performed; current densities of 500 mu A cm(-2) were obtained in unstirred solutions

Characterization of Nanoporous Gold Electrodes for Bioelectrochemical Applications

The high surface areas of nanostructured electrodes can provide for significantly enhanced surface loadings of electroactive materials. The fabrication and characterization of nanoporous gold (np-Au) substrates as electrodes for bioelectrochemical applications is described. Robust np-Au electrodes were prepared by sputtering a gold–silver alloy onto a glass support and subsequent dealloying of the silver component. Alloy layers were prepared with either a uniform or nonuniform distribution of silver and, post dealloying, showed clear differences in morphology on characterization with scanning electron microscopy. Redox reactions under kinetic control, in particular measurement of the charge required to strip a gold oxide layer, provided the most accurate measurements of the total electrochemically addressable electrode surface area, <i>A</i>_real. Values of <i>A</i>_real up to 28 times that of the geometric electrode surface area, <i>A</i>_geo, were obtained. For diffusion-controlled reactions, overlapping diffusion zones between adjacent nanopores established limiting semi-infinite linear diffusion fields where the maximum current density was dependent on <i>A</i>_geo. The importance of measuring the surface area available for the immobilization was determined using the redox protein, <i>cyt</i> c. The area accessible to modification by a biological macromolecule, <i>A</i>_macro, such as <i>cyt</i> c was reduced by up to 40% compared to <i>A</i>_real, demonstrating that the confines of some nanopores were inaccessible to large macromolecules due to steric hindrances. Preliminary studies on the preparation of np-Au electrodes modified with osmium redox polymer hydrogels and Myrothecium verrucaria bilirubin oxidase (<i>Mv</i>BOD) as a biocathode were performed; current densities of 500 μA cm^–2 were obtained in unstirred solutions

Mediated electron transfer of cellobiose dehydrogenase and glucose oxidase at osmium polymer modified nanoporous gold electrodes.

Nanoporous and planar gold electrodes were utilised as supports for the redox enzymes Aspergillus niger glucose oxidase (GOx) and Corynascus thermophilus cellobiose dehydrogenase (CtCDH). Electrodes modified with hydrogels containing enzyme, Os-redox polymers and the cross-linking agent poly(ethylene glycol)diglycidyl ether (PEGDGE) were used as biosensors for the determination of glucose and lactose. Limits of detection of 6.0 (± 0.4), 16.0 (± 0.1) and 2.0 (± 0.1) μM were obtained for CtCDH modified lactose and glucose biosensors and GOx modified glucose biosensors, respectively, at nanoporous gold electrodes. Biofuel cells comprised of GOx and CtCDH modified gold electrodes were utilised as anodes, together with Myrothecium verrucaria bilirubin oxidase (MvBOD) or Melanocarpus albomyces laccase (rMaLc) as cathodes, in biofuel cells. A maximum power density of 41 μW/cm2 was obtained for a CtCDH/MvBOD biofuel cell in 5 mM lactose and O2 saturated buffer (pH 7.4, 0.1 M phosphate, 150 mM NaCl)

Further Insights into the Catalytical Properties of Deglycosylated Pyranose Dehydrogenase from Agaricus meleagris Recombinantly Expressed in Pichia pastoris

The present study focuses on fragmented deglycosylated pyranose dehydrogenase (fdgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris. Fragmented deglycosylated PDH is formed from the deglycosylated enzyme (dgPDH) when it spontaneously loses a C-terminal fragment when stored in a buffer solution at 4 °C. The remaining larger fragment has a molecular weight of ∼46 kDa and exhibits higher volumetric activity for glucose oxidation compared with the deglycosylated and glycosylated (gPDH) forms of PDH. Flow injection amperometry and cyclic voltammetry were used to assess and compare the catalytic activity of the three investigated forms of PDH, “wired” to graphite electrodes with two different osmium redox polymers: [Os­(4,4′-dimethyl-2,2′-bipyridine)₂(poly­(vinylimidazole))₁₀Cl]⁺ [Os­(dmbpy)­PVI] and [Os­(4,4′-dimethoxy-2,2′-bipyridine)₂(poly-(vinylimidazole))₁₀Cl]⁺ [Os­(dmobpy)­PVI]. When “wired” with Os­(dmbpy)­PVI, the graphite electrodes modified with fdgPDH showed a pronounced increase in the current density with <i>J</i>_max 13- and 6-fold higher than that observed for gPDH- and dgPDH-modified electrodes, making the fragmented enzyme extraordinarily attractive for further biotechnological applications. An easier access of the substrate to the active site and improved communication between the enzyme and mediator matrix are suggested as the two main reasons for the excellent performance of the fdgPDH when compared with that of gPDH and dgPDH. Three of the four glycosites in PDH: N⁷⁵, N¹⁷⁵, and N²⁵² were assigned using mass spectrometry in conjunction with endoglycosidase treatment and tryptic digestion. Determination of the asparagine residues carrying carbohydrate moieties in PDH can serve as a solid background for production of recombinant enzyme lacking glycosylation