Analyses of the Large Subunit Histidine-Rich Motif Expose an Alternative Proton Transfer Pathway in [NiFe] Hydrogenases

A highly conserved histidine-rich region with unknown function was recognized in the large subunit of [NiFe] hydrogenases. The HxHxxHxxHxH sequence occurs in most membrane-bound hydrogenases, but only two of these histidines are present in the cytoplasmic ones. Site-directed mutagenesis of the His-rich region of the T. roseopersicina membrane-attached Hyn hydrogenase disclosed that the enzyme activity was significantly affected only by the replacement of the His104 residue. Computational analysis of the hydrogen bond network in the large subunits indicated that the second histidine of this motif might be a component of a proton transfer pathway including Arg487, Asp103, His104 and Glu436. Substitutions of the conserved amino acids of the presumed transfer route impaired the activity of the Hyn hydrogenase. Western hybridization was applied to demonstrate that the cellular level of the mutant hydrogenases was similar to that of the wild type. Mostly based on theoretical modeling, few proton transfer pathways have already been suggested for [NiFe] hydrogenases. Our results propose an alternative route for proton transfer between the [NiFe] active center and the surface of the protein. A novel feature of this model is that this proton pathway is located on the opposite side of the large subunit relative to the position of the small subunit. This is the first study presenting a systematic analysis of an in silico predicted proton translocation pathway in [NiFe] hydrogenases by site-directed mutagenesis

Widespread applicability of bacterial cellulose-ZnO-MWCNT hybrid membranes

The novel photoactive membranes have grabbed the attention in the field of environmental protection by employing wastewater treatments and the removal of microorganisms or organic pollutants from wastewater. Here we present a promising self-supported photoactive hybrid membrane for future antimicrobial and water treatment applications. In this study, the efficiency of bacterial cellulose (BC) - zinc oxide (ZnO) - multi walled carbon nanotube (MWCNT) hybrid membranes in the adsorption and photocatalytic degradation of methylene blue (MB) under UV radiation and the removal of Escherichia coli (E. coli) was investigated. It was found that the photocatalytic efficiency is strongly dependent on both the preparation method and the amount of ZnO-MWCNT additives loaded into the hybrid membranes. The characterization of BC-ZnO-MWCNT membranes was done using scanning electron microscopy (SEM), mercury intrusion porosimetry (MIP), and X-ray micro computed tomography (μCT) to study the morphological and porosity aspect of the prepared-membranes. The promising results of this study could provide a new pathway in the field of photocatalysed-based water treatment technology by the application of hybrid membranes

A Simplified and Efficient Method for Production of Manganese Ferrite Magnetic Nanoparticles and Their Application in DNA Isolation

A simplified, fast, and effective production method has been developed for the synthesis of manganese ferrite (MnFe2O4) magnetic nanoparticles (MNPs). In addition to the wide applicability of MnFe2O4 MNPs, this work also reports their application in DNA isolation for the first time. An ultrasonic-cavitation-assisted combustion method was applied in the synthesis of MnFe2O4 MNPs at different furnace temperatures (573 K, 623 K, 673 K, and 773 K) to optimize the particles’ properties. It was shown that MnFe2O4 nanoparticles synthesized at 573 K consist of a spinel phase only with adequate size and zeta potential distributions and superparamagnetic properties. It was also demonstrated that superparamagnetic manganese ferrite nanoparticles bind DNA in buffer with a high NaCl concentration (2.5 M), and the DNA desorbs from the MNPs by decreasing the NaCl concentration of the elution buffer. This resulted in a DNA yield comparable to that of commercial DNA extraction products. Both the DNA concentration measurements and electrophoresis confirmed that a high amount of isolated bacterial plasmid DNA (pDNA) with adequate purity can be extracted with MnFe2O4 (573 K) nanoparticles by applying the DNA extraction method proposed in this article

A Simplified and Efficient Method for Production of Manganese Ferrite Magnetic Nanoparticles and Their Application in DNA Isolation

A simplified, fast, and effective production method has been developed for the synthesis of manganese ferrite (MnFe2O4) magnetic nanoparticles (MNPs). In addition to the wide applicability of MnFe2O4 MNPs, this work also reports their application in DNA isolation for the first time. An ultrasonic-cavitation-assisted combustion method was applied in the synthesis of MnFe2O4 MNPs at different furnace temperatures (573 K, 623 K, 673 K, and 773 K) to optimize the particles’ properties. It was shown that MnFe2O4 nanoparticles synthesized at 573 K consist of a spinel phase only with adequate size and zeta potential distributions and superparamagnetic properties. It was also demonstrated that superparamagnetic manganese ferrite nanoparticles bind DNA in buffer with a high NaCl concentration (2.5 M), and the DNA desorbs from the MNPs by decreasing the NaCl concentration of the elution buffer. This resulted in a DNA yield comparable to that of commercial DNA extraction products. Both the DNA concentration measurements and electrophoresis confirmed that a high amount of isolated bacterial plasmid DNA (pDNA) with adequate purity can be extracted with MnFe2O4 (573 K) nanoparticles by applying the DNA extraction method proposed in this article

<i>In vivo</i> and <i>in vitro</i> hydrogenase activity of <i>Thiocapsa roseopersicina</i> HynSL enzyme.

<p><i>In vivo</i> and <i>in vitro</i> hydrogenase activity of <i>Thiocapsa roseopersicina</i> HynSL enzyme.</p

Bacterial strains.

<p>Indicated strains and plasmids are from Stratagene, La Jolla, CA, USA.</p

Mutagenesis primers used for creating new mutants.

<p>Nucleotide triplets coding mutated amino acids are bold.</p

Arrangement of conserved histidines.

<p>T.r.: <i>Thiocapsa roseopersicina</i> HynL, A.v.: <i>Allochromatium vinosum</i> HydL, R.c.: <i>Rhodobacter capsulatus</i> HupL, M.c.: <i>Methylococcus capsulatus</i>, B.j.: <i>Bradyrhizobium japonicum</i> HupL, Rh.l.: <i>Rhizobium leguminosarum</i> HupL, R.e.: <i>Ralstonia eutropha</i> H16 HoxG, D.v.: <i>Desulfovibrio vulgaris</i> Miyazaki F HynB, D.g.: <i>Desulfovibrio gigas</i> HynB, D.f.: <i>Desulfovibrio fructosovorans</i> HynB. The numbering refers to the <i>T. roseopersicina</i> HynL subunit.</p

The amino acids of proposed proton transfer pathway are highly conserved.

<p>T.r.: <i>Thiocapsa roseopersicina</i> HynL, A.v.: <i>Allochromatium vinosum</i> HydL, R.c.: <i>Rhodobacter capsulatus</i> HupL, M.c.: <i>Methylococcus capsulatus</i>, B.j.: <i>Bradyrhizobium japonicum</i> HupL, Rh.l.: <i>Rhizobium leguminosarum</i> HupL, R.e.: <i>Ralstonia eutropha</i> H16 HoxG, D.v.: <i>Desulfovibrio vulgaris</i> Miyazaki F HynB, D.g.: <i>Desulfovibrio gigas</i> HynB, D.f.: <i>Desulfovibrio fructosovorans</i> HynB. The upper and lower numbering refers to the large subunits of <i>T. roseopersicina</i> and <i>D. vulgaris</i> enzymes, respectively.</p