Production and purification of a soluble hydrogenase from Ralstonia eutropha H16 for potential hydrogen fuel cell applications

Bat-Erdene Jugder; Helene Lebhar; Kondo-Francois Aguey-Zinsou; Christopher P Marquis

doi:10.1016/j.mex.2016.03.005

Production and purification of a soluble hydrogenase from Ralstonia eutropha H16 for potential hydrogen fuel cell applications

MethodsX. 2016 Mar 22:3:242-50. doi: 10.1016/j.mex.2016.03.005. eCollection 2016.

Authors

Bat-Erdene Jugder¹, Helene Lebhar¹, Kondo-Francois Aguey-Zinsou², Christopher P Marquis¹

Affiliations

¹ School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052 Australia.
² MERLin group, School of Chemical Engineering, University of New South Wales, Sydney 2052 Australia.

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.