*** 9623832 Steffan This Small Business Innovation Research Phase II project is aimed at producing cost-effective biological catalysts for use in the bioremediation of chlorinated solvent-contaminated environments. Toluene-4-monooxygenase (T4MO) is a multicomponent enzyme from Pseudomonas mendocina KRl capable of oxidizing chlorinated solvents, which directly leads to their bioremediation. During Phase I, several regions of amino acid sequence were identified that control the reactivity of T4MO. In vitro mutagenic techniques (either site-directed or random primed polymerase chain reaction) were then used to alter the amino acid sequence in these regions. This effort resulted in the creation of new, catalytically-active mutant isoforms of T4MO. One of the most promising of these new isoforms has an increased oxidation rate for halogenated hydrocarbons such as trichloroethylene (TCE) and chloroform, while several other isoforms exhibit changes in substrate specificity relative to the wild-type enzyme. These results demonstrate the soundness of the project's strategy to improve the catalytic versatility of the T4MO enzyme via genetic engineering. Both of these mutant isoforms may prove valuable in commercial applications. The proposed Phase II project will evaluate the commercial potential of these promising mutants in detail, including further characterization of oxidation rates, substrate specificity ranges, product distributions, catalyst stability, bioreactor scale-up parameters, and other relevant structural and functional properties of these novel, engineered biocatalysts. The biocatalysts developed during this project will be commercially applicable to remediate many of the Nation's contaminated aquifers and soils at both government and private sites. Initial application will be in contained vapor-phase bioreactors, and future applications will include in situ treatment. ***
