Purification and characterization of [Fe]-hydrogenase from high yielding hydrogen-producing strain, Enterobacter cloacae IIT-BT08 (MTCC 5373)

Fe-hydrogenase from Enterobacter cloacae IIT-BT08 was purified 1284 fold with specific activity of 335 μmol H2/min/mg protein for hydrogen evolution using reduced methyl viologen as an electron-donor at 25 °C. The molecular weight of the monomeric enzyme was determined to be 51 kDa by MALDI-ToF mass spectrometry. The PI of the enzyme was ∼5.6 displaying its acidic nature. The optimal temperature and pH for hydrogen evolution was 37 °C and 7–7.2 respectively. The affinity constant, Km for reduced methyl viologen was 0.57 ± 0.03 mM and that of reduced ferredoxin was 0.72 ± 0.04 μM. The enzyme contained ∼11.47 gm-atom Fe/mol of Fe-hydrogenase. Electron paramagnetic resonance analysis ascertained the existence of iron molecules as [4Fe–4S] clusters. The internal amino acid sequences of trypsin digested peptides of hydrogenase as determined by ESI MS/MS Q-ToF showed 80-87% identities with the respective sequences of Clostridium sp. and Trichomonas sp. hydrogenase.