Isolation and molecular characterization of hydrogenase gene from a high rate of hydrogen-producing bacterial strain Enterobacter cloacae IIT-BT 08

Degenerate PCR primers (forward as well as the reverse) were designed from the protein sequences of known structural genes encoding the catalytic subunits of Fe-hydrogenases obtained from NCBI Protein Sequence Database. Amino acid sequences were multiple aligned through CLUSTAL-W software and compared for conserved sequence motifs. Mole percent of G+C of the present strain is found to be 55% by thermal melting method. An amplified PCR product of 1 kb in size was obtained from the genomic DNA of Enterobacter cloacae IIT-BT 08 by using a set of (JM-1 and JM-12) degenerate primer. Gel purified PCR product was cloned in TOPO TA cloning vector (3.9 kb) and transformed into TOPOF’ cells. Initial experiment on Southern hybridization of the PCR product showed no homology with hydA gene of Clostridium acetobutylicum ATCC 824.