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

The soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising candidate enzyme for H(2)-based biofuel application as it favours H(2) 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 H(2) oxidizing activity of the SH was demonstrated spectrophotometrically in solution and also immobilized on an EPG electrode using cyclic voltammetry.