Purification and characterization of putative alkaline [Ni–Fe] hydrogenase from unicellular marine green alga, Tetraselmis kochinensis NCIM 1605

SummaryHydrogenase enzyme from the unicellular marine green alga Tetraselmis kochinensis NCIM 1605 was purified 467 fold to homogeneity. The molecular weight was estimated to be ∼89 kDa by SDS–PAGE. This enzyme consists of two subunits with molecular masses of ∼70 and ∼19 kDa. The hydrogenase was found to contain 10 g atoms of Fe and 1 g of atom of Ni per mole of protein. The specific activity of hydrogen evolution was 50 μmol H2/mg/h of enzyme using reduced methyl viologen as an electron donor. This hydrogenase enzyme has pI value ∼9.6 representing its alkaline nature. The absorption spectrum of the hydrogenase enzyme showed an absorption peak at 425 nm indicating that the enzyme had iron–sulfur clusters. The total of 16 cysteine residues were found per mole of enzyme under the denaturing condition and 20 cysteine residues in reduced denatured enzyme indicating that it has two disulfide bridges.