Molecular cloning and characterization of the gene coding for azoreductase from Bacillus sp. OY1-2 isolated from soil

J Biol Chem. 2001 Mar 23;276(12):9059-65. doi: 10.1074/jbc.M008083200. Epub 2000 Dec 27.


Azo dyes are regarded as pollutants because they are not readily reduced under aerobic conditions. Bacillus sp. OY1-2 transforms azo dyes into colorless compounds, and this reduction is mediated by a reductase activity for the azo group in the presence of NADPH. A 1.2-kbp EcoRI fragment containing the gene that encodes azoreductase was cloned by screening the genomic library of Bacillus sp. OY1-2 with digoxigenin-labeled probe designed from the N-terminal amino acid sequence of the purified enzyme. An open reading frame encoding the azoreductase, consisting of 178 amino acids, was predicted from the nucleotide sequence. In addition, because only a Bacillus subtillis hypothetical protein was discovered in the public databases (with an amino acid identity of 52.8%), the gene encoding the azoreductase cloned in this study was predicted to be a member of a novel family of reductases. Southern blot analysis revealed that the azoreductase gene exists as a single copy gene on a chromosome. Escherichia coli-expressing recombinant azoreductase gave a ten times greater reducing activity toward azo dyes than the original Bacillus sp. OY1-2. In addition, the expressed azoreductase purified from the recombinant E. coli lysate by Red-Sepharose affinity chromatography showed a similar activity and specificity as the native enzyme. This is the first report describing the sequencing and characterization of a gene encoding the azo dye-reducing enzyme, azoreductase, from aerobic bacteria and its expression in E. coli.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacillus / enzymology
  • Bacillus / genetics*
  • Bacillus / isolation & purification
  • Base Sequence
  • Cloning, Molecular
  • DNA, Bacterial
  • Escherichia coli / genetics
  • Molecular Sequence Data
  • NADH, NADPH Oxidoreductases / chemistry
  • NADH, NADPH Oxidoreductases / genetics*
  • NADH, NADPH Oxidoreductases / isolation & purification
  • Nitroreductases
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Sequence Homology, Amino Acid
  • Soil Microbiology*


  • DNA, Bacterial
  • Recombinant Proteins
  • NADH, NADPH Oxidoreductases
  • Nitroreductases
  • azoreductase

Associated data

  • GENBANK/AB002631