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Journal of Bacteriology, January 2002, p. 344-349, Vol. 184, No. 2
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.2.344-349.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Directed Evolution of Toluene ortho-Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation

Keith A. Canada, Sachiyo Iwashita,,{dagger} Hojae Shim,,{dagger} and Thomas K. Wood*

Departments of Chemical Engineering and Molecular & Cellular Biology, University of Connecticut, Storrs, Connecticut 06269-3222

Received 13 September 2001/ Accepted 15 October 2001

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.

* Corresponding author. Mailing address: Departments of Chemical Engineering and Molecular & Cellular Biology, University of Connecticut, Storrs, CT 06269-3222. Phone: (860) 486-2483. Fax: (860) 486-2959. E-mail: twood{at}engr.uconn.edu.

{dagger} Present address: Department of Civil & Environmental Engineering, Hanyang University, Kyungkido 425-791, Korea.

Journal of Bacteriology, January 2002, p. 344-349, Vol. 184, No. 2
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.2.344-349.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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