Directed evolution of toluene ortho-monooxygenase for enhanced 1-naphthol synthesis and chlorinated ethene degradation

K. A. Canada, S. Iwashita, H. Shim, T. K. Wood

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Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chemical manufacturing intermediate. Directed evolution was used to increase the activity of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 for both chlorinated ethenes and naphthalene oxidation. When expressed in Escherichia coli, the variant TOM-Green degraded TCE (2.5 ± 0.3 versus 1.39 ± 0.05 nmol/min/mg of protein), 1,1-dichloroethylene, and trans-dichloroethylene more rapidly. Whole cells expressing TOM-Green synthesized 1-naphthol at a rate that was six times faster than that mediated by the wild-type enzyme at a concentration of 0.1 mM (0.19 ± 0.03 versus 0.029 ± 0.004 nmol/min/mg of protein), whereas at 5 mM, the mutant enzyme was active (0.07 ± 0.03 nmol/min/mg of protein) in contrast to the wild-type enzyme, which had no detectable activity. The regiospecificity of TOM-Green was unchanged, with greater than 97% 1-naphthol formed. The beneficial mutation of TOM-Green is the substitution of valine to alanine in position 106 of the α-subunit of the hydroxylase, which appears to act as a smaller "gate" to the diiron active center. This hypothesis was supported by the ability of E. coli expressing TOM-Green to oxidize the three-ring compounds, phenanthrene, fluorene, and anthracene faster than the wild-type enzyme. These results show clearly that random, in vitro protein engineering can be used to improve a large multisubunit protein for multiple functions, including environmental restoration and green chemistry.

Original languageEnglish (US)
Pages (from-to)344-349
Number of pages6
JournalJournal of bacteriology
Issue number2
StatePublished - 2002

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology


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