Directed Evolution of Toluene Ortho-Monooxygenase for Enhanced 1-naphthol Synthesis and Chlorinated Ethene Degradation

J Bacteriol. 2002 Jan;184(2):344-9. doi: 10.1128/jb.184.2.344-349.2002.


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 alpha-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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Burkholderia cepacia / enzymology
  • Burkholderia cepacia / genetics
  • Dichloroethylenes / metabolism*
  • Directed Molecular Evolution
  • Gene Library
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / metabolism*
  • Mutagenesis
  • Naphthols / metabolism*
  • Sequence Homology, Nucleic Acid
  • Substrate Specificity
  • Toluene / metabolism
  • Trichloroethylene / metabolism*


  • Dichloroethylenes
  • Naphthols
  • vinylidene chloride
  • Trichloroethylene
  • 1-naphthol
  • Toluene
  • Mixed Function Oxygenases
  • toluene ortho-monooxygenase