Purification and Characterization of Hydrogenase from Ethanoligenens harbinense YUAN-3

Hydrogenase is the key terminal enzyme in electron delivery within hydrogen metabolism in bacteria, which has been attracted many attentions. In the present study, crude enzyme was firstly prepared by cell broken. Then a kind of hydrogenase from Ethanoligenens harbinense YUAN-3 was purified by chromatography methods on Sephadex G-100 and DEAE 52 columns. The enzyme was purified 169-fold with 7.8% recovery of activity, resulting in a specific activity for hydrogen evolution of 41.6 μmol/min/mg of protein, using reduced methyl viologen as an electron donor. The purity of the enzyme was judged by native PAGE. The molecular weight was estimated to be 60 kDa by SDS-PAGE. The purification procedures and parameters was the first report on hydrogenase from YUAN-3.