Cloning, sequencing and expression of the gene encoding NADH oxidase from the extreme anaerobic thermophile Thermoanaerobium brockii

Abstract

The gene encoding the enzyme NADH oxidase from the extreme thermophile Thermoanaerobium brockii has been isolated from a recombinant library of genomic DNA and sequenced. An open reading frame corresponds to the 651 amino acids of the enzyme's subunit, which include characteristic FAD- and NADH-binding sequences, as well as cysteines which are involved in the FeS cluster present in the enzyme. The enzyme is expressed either from its own promoter or from vector promoters in Escherichia coli. After heat-treating the recombinant extracts at 70 degrees C, most of the host proteins are denatured, leaving the NADH oxidase 5- to 10-fold enriched.