Detection and amplification of putative [NiFe] hydrogenase operon in anoxybacillus sp. SK3-4

Abstract

Most fuel cells nowadays use platinum as the electrocatalyst, but this metal is expensive and difficult to be obtained. This gives rise to many studies on enzymatic fuel cells. The major hurdle in enzymatic fuel cell is that the hydrogen oxidation at anode is sensitive to oxygen, and this requires an oxygen-insensitive enzyme as the catalyst. [NiFe] hydrogenase, comprised of two subunits, is the best known hydrogenase to be used as biocatalyst since it is insensitive to oxygen. [NiFe] hydrogenase genes are organized in an operon, containing a cluster of genes needed for the maturation of the enzyme. This study was initiated to detect and amplify the genes in putative [NiFe] hydrogenase operon in Anoxybacillus sp. SK3-4 by PCR and DNA sequencing. The sequences of the amplified genes were comfirmed by alignment with the published sequences in the NCBI database. 11 pairs of primer were designed for both PCR and sequencing, based on the full genome sequence of Anoxybacillus sp. SK3-4. All the primers designed are 18-22 nucleotides in length with a minimum GC content of 50%. The difference in Tm between the primers pairs are in the range of 0.1-2.3oC. It can be comfirmed that the putative [NiFe] hydrogenase operon consists of 11 genes that are transcribed into one polycistronic mRNA with 10 101 nucleotides and translated into 11 different proteins. Futher sequence analysis showed that [NiFe] hydrogenase in Anoxybacillus sp. SK3-4 is predicted to be classified into group 1 hydrogenase, which is a membrane-bound hydrogenase. The large subunit protein is the most important protein in the [NiFe] hydrogenase operon where the catalytic activity resdes. The large subunit has 57% similarity with [NiFe] hydrogenase from Allochromatum Vinosum (PDB ID 3MYR). A homology model has been constructed for Anoxybacillus sp. SK3-4 [NiFe] hydrogenase using the template structure of A. vinosum. The large subunit consist of 1720 nucleotides that codes for 573 amino acids with a molecular weight of 64.7 kDa. The theoritical pI has been predicted to be 6.25.