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 ␣-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.

The metal-forming industries require the use of greases to lubricate metal surfaces during manufacturing operations, and the residues of these lubricants must be removed prior to finishing processes to protect and improve the appearance of the final product. An aqueous, biological metal-cleaning process operating under mild conditions (pH 9, 42 degrees C) eliminates the use of environmentally unfriendly cleaning materials such as chlorinated solvents by employing microorganisms to degrade greases and oils naturally. This process was characterized in terms of initial degradation rates of a representative metal lubricant and by phylogenetic identification of the active bacteria. The metal lubricant in a surfactant solution was degraded by a bacterial consortium, and its concentration was determined by a novel gas chromatography assay. The maximum degradation rate Vmax and the apparent Km were obtained as 45 mg/(day mg protein) and 24 g/l on cellular basis, and 4.6 g/(day l) and 33 g/l on a volumetric basis, respectively. Mineralization of the metal lubricant was shown by analyzing the evolved CO2 and Cl-, and the bacterial consortium utilized the metal lubricant as a sole carbon and energy source (micro=0.05+/-0.01 h(-1) at 0.5 vol% lubricant concentration). The active bacteria in the biological metal-cleaning process were identified as Bacillus licheniformis for the higher lubricant concentrations (3, 5, and 7.5 vol%), Bacillus cereus at 1 vol%, and Pseudomonas aeruginosa, Rhizobiaceae strain M100, and Achromobacter sp. LMG 5431 at 0.3 vol%.