We studied the consequences of point mutations in 2NTDO on the substrate spectrum of the enzymes. To that end, we compared oxygenation efficiencies of 2NTDO and five variants from the 4NT + and 3NT + experiments with the four substrates, nitrobenzene, and the three nitrotoluene isomers. ants and the substrate turnover in these enzyme assays was mostly insufficient to allow reliable quantification of f O 2 -uc close to unity. We note that the serial enrichment cultures in the 4NT + experiments ran for 6 months as compared to only one month in the 3NT + experiments. Whether this 6-fold shorter adaptation period in the 3NT + experiments is responsible for the more exclusive adaptation towards this one substrate is unclear.