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Production and Purification of a Soluble Hydrogenase From Ralstonia Eutropha H16 for Potential Hydrogen Fuel Cell Applications

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Production and Purification of a Soluble Hydrogenase From Ralstonia Eutropha H16 for Potential Hydrogen Fuel Cell Applications

Bat-Erdene Jugder et al. MethodsX.

Abstract

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.

Keywords: Cupriavidus necator; Hydrogen oxidation; Purification; Ralstonia eutropha; Soluble hydrogenase; Soluble hydrogenase purification from Ralstonia eutropha.

Figures

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Fig. 1
Fig. 1
Purification of the soluble hydrogenase (SH). A) Q Sepharose FF chromatogram, stepwise gradients. SH active fractions were eluted in the first peak (Abs at 280 nm) during a linear gradient of 0–43% B. B) RESOURCE Q chromatogram, stepwise gradients. SH active fractions were eluted in the first peak (shadowed) during a linear gradient of 0–20% B. C1) Superdex 200 chromatogram. 50 mM KPi buffer supplemented with 150 mM NaCl.
Fig. 2
Fig. 2
SDS-PAGE of the pure soluble hydrogenase. Lane 1: SeeBlue® Plus2 Pre-Stained Standard; Lane 2: The SH fraction from the 50 mM KPi buffer with 150 mM NaCl gel filtration; 10 μg protein was loaded into the well. The hexameric SH comprises of 5 subunits; Hox I (two copies), HoxY, HoxU, HoxH, Hox F.
Fig. 3
Fig. 3
Cyclic voltammograms for pure SH immobilized on an EPG electrode. A) H2 oxidation in the presence and absence of Polymyxin with SH. Both electrodes were activated by NADH. 5 μL of activated SH only (a in blue) and mixture of 3 μL of activated hydrogenase and 2 μL of 2% of Polymyxin (b in red) are shown. B) H2 oxidation following different incubation times at an applied potential of −550 mV (vs. NHE). 5 μL of SH was applied on an EPG electrode. 5 min (a in pink), 10 min (b in green) and 30 min (c in blue). All cyclic voltametric experiments were carried out in 50 mM KPi buffer (pH 7.0) at a scan rate 50 mV s−1.I − Current, E − potential, NHE − Normal Hydrogen Electrode. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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