Vitreoscilla hemoglobin enhances the first step in 2,4-dinitrotoluene degradation in vitro and at low aeration in vivo

The initial enzyme in 2,4-dinitrotoluene (2,4-DNT) catabolism from Burkhold eria sp. strain DNT is DNT dioxygenase, which catalyzes oxygen addition to 2,4-DNT to form 4-methyl-5-nitrocatechol (MNC). The gene for this enzyme, d ntA, was placed into Escherichia call both in the presence and absence of t he Vitreoscilla hemoglobin gene, vgb, producing strains PF6 and PFJS39, res pectively. PF6 outgrew PFJS39 in LB medium and at restricted aeration in mi nimal medium containing 110 mu M (20 ppm) 2,4-DNT. When grown in minimal me dium containing 110 mu M 2,4-DNT with normal aeration, the two strains conv erted 2,4-DNT to MNC at almost the same rate, while with restricted aeratio n the rate for PF6 was twice that of PFJS39, The V-max and K-M for 2,4-DNT for the conversion was determined using whole cells and whole cell lysates of both strains. For cells grown under both normal and restricted aeration in minimal/110 mu M 2,4-DNT medium there was a three-to four-fold increase in V-max for PF6 compared to PFJS39 but also about a 50% increase in the ap parent K-M. These results support the idea that Vitreoscilla hemoglobin can enhance bioremediation pathways of aromatic compounds that require oxygen addition at one or more steps. (C) 2000 Elsevier Science B.V. All rights re served.