Saturation mutagenesis of 2,4-DNT dioxygenase of Burkholderia sp. strain DNT for enhanced dinitrotoluene degradation

Thammajun Leungsakul, Brendan G. Keenan, Hong Yin, Barth F. Smets, Thomas K. Wood

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Abstract

2,4-Dinitrotoluene (2,4-DNT) and 2,6-DNT are priority pollutants, and 2,4-DNT dioxygenase of Burkholderia sp. strain DNT (DDO) catalyzes the initial oxidation of 2,4-DNT to form 4-methyl-5-nitrocatechol and nitrite but has significantly less activity on other dinitrotoluenes and nitrotoluenes (NT). Hence, oxidation of 2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 2NT, and 4NT were enhanced here by performing saturation mutagenesis on codon 1204 of the α subunit (DntAc) of DDO and by using a membrane agar plate assay to detect catechol formation. Rates of degradation were quantified both by the formation of nitrite and by the formation of the intermediates with high performance liquid chromatography. The degradation of both 2,3-DNT and 2,5-DNT were achieved for the first time (no detectable activity with the wild-type enzyme) using whole Escherichia coli TG1 cells expressing DDO variants DntAc I204L and I204Y (0.70 ± 0.03 and 0.22 ± 0.02 nmol/min/mg protein for 2,5-DNT transformation, respectively). DDO DntAc variant I204L also transformed both 2,6-DNT and 2,4-DNT 2-fold faster than wild-type DDO (0.8 ± 0.6 nmol/min/mg protein and 4.7 ± 0.5 nmol/min/mg protein, respectively). Moreover, the activities of DDO for 2NT and 4NT were also enhanced 3.5-fold and 8-fold, respectively. Further, DntAc variant I204Y was also discovered with comparable rate enhancements for the substrates 2,4-DNT, 2,6-DNT, and 2NT but not 4NT. Sequencing information obtained during this study indicated that the 2,4-DNT dioxygenases of Burkholderia sp. strain DNT and B. cepacia R34 are more closely related than originally reported. This is the first report of engineering an enzyme for enhanced degradation of nitroaromatic compounds and the first report of degrading 2,5-DNT.

Original languageEnglish (US)
Pages (from-to)416-426
Number of pages11
JournalBiotechnology and bioengineering
Volume92
Issue number4
DOIs
StatePublished - Nov 20 2005

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All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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