Production and purification of a soluble hydrogenase from Ralstonia eutropha H16 for potential hydrogen fuel cell applications
نویسندگان
چکیده
The soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising candidate enzyme for H2-based biofuel application as it favours H2 oxidation and is relatively oxygen-tolerant. In this report, bioprocess development studies undertaken to produce and purify an active SH are described, based on the methods previously reported [1], [2], [3], [4]. Our modifications are: •Upstream method optimizations were undertaken on heterotrophic growth media and cell lysis involving ultrasonication.•Two anion exchangers (Q Sepharose and RESOURCE Q) and size exclusion chromatographic (Superdex 200) matrices were successfully employed for purification of a hexameric SH from R. eutropha.•The H2 oxidizing activity of the SH was demonstrated spectrophotometrically in solution and also immobilized on an EPG electrode using cyclic voltammetry.
منابع مشابه
An analysis of the changes in soluble hydrogenase and global gene expression in Cupriavidus necator (Ralstonia eutropha) H16 grown in heterotrophic diauxic batch culture
BACKGROUND Soluble hydrogenases (SH) are enzymes that catalyse the oxidation of molecular hydrogen. The SH enzyme from Cupriavidus necator H16 is relatively oxygen tolerant and makes an attractive target for potential application in biochemical hydrogen fuel cells. Expression of the enzyme can be mediated by derepression of the hox promoter system under heterotrophic conditions. However, the ov...
متن کاملIncreasing the metabolic capacity of Escherichia coli for hydrogen production through heterologous expression of the Ralstonia eutropha SH operon
BACKGROUND Fermentative hydrogen production is an attractive means for the sustainable production of this future energy carrier but is hampered by low yields. One possible solution is to create, using metabolic engineering, strains which can bypass the normal metabolic limits to substrate conversion to hydrogen. Escherichia coli can degrade a variety of sugars to hydrogen but can only convert e...
متن کاملA hydrogen-sensing multiprotein complex controls aerobic hydrogen metabolism in Ralstonia eutropha.
H(2) is an attractive energy source for many microorganisms and is mostly consumed before it enters oxic habitats. Thus aerobic H(2)-oxidizing organisms receive H(2) only occasionally and in limited amounts. Metabolic adaptation requires a robust oxygen-tolerant hydrogenase enzyme system and special regulatory devices that enable the organism to respond rapidly to a changing supply of H(2). The...
متن کاملElectrocatalytic hydrogen oxidation by an enzyme at high carbon monoxide or oxygen levels.
Use of hydrogen in fuel cells requires catalysts that are tolerant to oxygen and are able to function in the presence of poisons such as carbon monoxide. Hydrogen-cycling catalysts are widespread in the bacterial world in the form of hydrogenases, enzymes with unusual active sites composed of iron, or nickel and iron, that are buried within the protein. We have established that the membrane-bou...
متن کاملHydrogen-driven asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol by Ralstonia eutropha transformant expressing alcohol dehydrogenase from Kluyveromyces lactis
BACKGROUND Conversion of industrial processes to more nature-friendly modes is a crucial subject for achieving sustainable development. Utilization of hydrogen-oxidation reactions by hydrogenase as a driving force of bioprocess reaction can be an environmentally ideal method because the reaction creates no pollutants. We expressed NAD-dependent alcohol dehydrogenase from Kluyveromyces lactis in...
متن کامل