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

l‑Arabinose Isomerase and d‑Xylose Isomerase from Lactobacillus reuteri: Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion of d‑Galactose and d‑Glucose

The l-arabinose isomerase (l-AI) and the d-xylose isomerase (d-XI) encoding genes from Lactobacillus reuteri (DSMZ 17509) were cloned and overexpressed in Escherichia coli BL21 (DE3). The proteins were purified to homogeneity by one-step affinity chromatography and characterized biochemically. l-AI displayed maximum activity at 65 °C and pH 6.0, whereas d-XI showed maximum activity at 65 °C and pH 5.0. Both enzymes require divalent metal ions. The genes were also ligated into the inducible lactobacillal expression vectors pSIP409 and pSIP609, the latter containing a food grade auxotrophy marker instead of an antibiotic resistance marker, and the l-AI- and d-XI-encoding sequences/genes were coexpressed in the food grade host Lactobacillus plantarum. The recombinant enzymes were tested for applications in carbohydrate conversion reactions of industrial relevance. The purified l-AI converted d-galactose to d-tagatose with a maximum conversion rate of 35%, and the d-XI isomerized d-glucose to d-fructose with a maximum conversion rate of 48% at 60 °C

Active-site geometry of pyranose oxidase from <i>T</i>. <i>multicolor</i>.

<p>In the closed form of the active-site loop of pyranose oxidase from <i>T</i>. <i>multicolor</i> (<i>Tm</i>POx, PDB code 1TT0; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.ref003" target="_blank">3</a>]), which is thought to be relevant for the oxidative half-reaction of POx [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.ref003" target="_blank">3</a>], Phe454 is positioned in the direct vicinity of the isoalloxazine ring and the C4a/N5 locus, at which oxygen is reduced. The figure was generated using PyMOL (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>).</p

Accessibility of methionine residues.

<p>Surface of the <i>T</i>. <i>multicolor</i> POx monomer in the vicinity of (<b>A</b>) Met417, showing the surface-exposed sulphur atom which is oxidised by H₂O₂, and (<b>B</b>) Met380 with its sulphur-containing side chain pointing towards the interior of the polypeptide matrix, in which it is buried and hence is not accessible from the surface.</p

Engineering Pyranose 2-Oxidase for Modified Oxygen Reactivity

<div><p>Pyranose 2-oxidase (POx), a member of the GMC family of flavoproteins, catalyzes the regioselective oxidation of aldopyranoses at position C2 to the corresponding 2-ketoaldoses. During the first half-reaction, FAD is reduced to FADH₂ and reoxidized in the second half-reaction by reducing molecular oxygen to H₂O₂. Alternative electron acceptors including quinones, radicals or chelated metal ions show significant and in some cases even higher activity. While oxygen as cheap and abundantly available electron acceptor is favored for many processes, reduced oxygen reactivity is desirable for some applications such as in biosensors/biofuel cells because of reduced oxidative damages to the biocatalyst from concomitant H₂O₂ production as well as reduced electron “leakage” to oxygen. The reactivity of flavoproteins with oxygen is of considerable scientific interest, and the determinants of oxygen activation and reactivity are the subject of numerous studies. We applied site-saturation mutagenesis on a set of eleven amino acids around the active site based on the crystal structure of the enzyme. Using microtiter plate screening assays with peroxidase/2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and 2,6-dichlorophenolindophenol, variants of POx with decreased oxidase activity and maintained dehydrogenase activity were identified. Variants T166R, Q448H, L545C, L547R and N593C were characterized with respect to their apparent steady-state constants with oxygen and the alternative electron acceptors DCPIP, 1,4-benzoquinone and ferricenium ion, and the effect of the mutations was rationalized based on structural properties.</p></div

Oxidation of methionines.

<p>Methionine-containing peptide fragments detected that show a mass increase of 16 or 32 Da after proteolytic digestion of <i>Tm</i>POx (inactivated during turnover of 100 mM D-glucose, treated with endogenous H₂O₂ or unaffected). Proteolytic digestion was performed with trypsin or Asp-N protease as indicated. The last column indicates whether the particular Met is considerably oxidised during substrate turnover and by H₂O₂ treatment compared to the unaffected sample (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.g002" target="_blank">Fig 2</a>).</p

Mass spectrometric identification of methionine residues oxidized by H₂O₂ during <i>Tm</i>POx inactivation.

<p>MALDI MS spectra were measured for unaffected POx (<b>A</b>), for POx inactivated during D-glucose oxidation (<b>B</b>) or for POx inactivated by endogenous H₂O₂ (<b>C</b>). The selected MALDI spectra in the left panel illustrate that Met497 of the tryptic peptide ITDAYNMPQPTFDFR with a theoretical MH⁺ of 1815.8 was extensively oxidised in <i>Tm</i>POx inactivated either during substrate turnover (<b>B</b>, left panel) or by H₂O₂ treatment (<b>C</b>, left panel). In contrast, some methionine residues were found not to be oxidised during <i>Tm</i>POx inactivation as shown for Met74 of the peptide VAMFDIGEIDSGLK having a MH⁺ of 1494.8 (right panel). The small signals at m/z 1510.8 are related to the oxidized form of the peptide generated due to the presence of air oxygen.</p

Identification of the exact site of oxidation in the peptide ⁴⁵²DAFSYGAVQQSIDSR⁴⁶⁶ by LC-ESI-MS/MS.

<p>The nearly complete series of C-terminal y-ions in the MS/MS spectrum of the double-charged ion of 830.4 confirms the peptide sequence. The +16 Da mass shift found for the ion y₁₃ further indicates that Phe454 is oxidised during substrate turnover and by H₂O₂ treatment.</p

Apparent steady-state kinetic constants of wild-type and mutant POx with D-glucose as electron donor (100 mM) and 1,4-BQ (varied from 0.01–0.5 mM) as electron acceptor.

<p>*data from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109242#pone.0109242-Spadiut2" target="_blank">[47]</a>.</p><p>Apparent steady-state kinetic constants of wild-type and mutant POx with D-glucose as electron donor (100 mM) and 1,4-BQ (varied from 0.01–0.5 mM) as electron acceptor.</p

Apparent steady-state kinetic constants of wild-type and mutant POx with D-glucose as electron donor (100 mM) and DCPIP (varied from 0.015–1.2 mM) as electron acceptor.

<p>Apparent steady-state kinetic constants of wild-type and mutant POx with D-glucose as electron donor (100 mM) and DCPIP (varied from 0.015–1.2 mM) as electron acceptor.</p