Burkholderia dinitrotoluene (DNT) dioxygenase in this study (from recombinant Esherichia coli strain PFJS39) is probably a multicomponent enzyme system that oxidizes 2,4-dinitrotoluene (DNT) to 4-methyl-5-nitrocatechol (MNC). DNT dioxygenase was purified by a four-step procedure that utilized consecutive gel filtration chromatography and a nondenaturing gel system. The purified enzyme oxidized DNT only in the presence of NADH and its yield increased by lipase pretreatment of crude cytosol. An estimated molecular weight of 100,000 was obtained by gel filtration. Polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) revealed the presence of three subunits for the samples from consecutive gel filtration chromatography and nondenaturing PAGE. Their molecular weights were 52,000-71,000, 23,000-25,500, and 12,000-16,500. These results suggest that DNT dioxygenase exists as a heterotrimer. The K_M of DNT dioxygenase for O₂ is 50 μ M, consistent with inhibition results of DNT dioxygenase by Vitreoscilla hemoglobin (its K_M for O₂ is 7 μ M). The K_M for DNT is 180 μ M. The purified enzyme is relatively stable below 40°C, retains activity over a broad pH range, and is stimulated by several cofactors in addition to NADH.

DOI: 10.1080/10889860903455378

Published in:  Bioremediation Journal, Volume 14, Issue 1 January 2010 , pages 38 - 53