This Small Business Innovative Research Phase I project involves the use of molecular biological techniques to alter the substrate specificity and activity of a biological catalysts for destroying a wide variety of recalcitrant chlorinated solvents. The putative active site of cloned toluene monooxygenase genes of Pseudomonas mendocina KR1 will be altered by using site directed mutagenesis so that it more closely resemble that of broad-substrate-specificity methane monooxygenases. The resulting catalysts will be tested for their ability to degrade a variety of chlorinated solvents heretofore not degraded by the wild-type organism. The effect of alterations in the putative oxygen binding region of the enzyme will also be examined. Chlorinated solvents are the most widespread and abundant contaminants of the Nation's groundwater and soils, and many are directly carcinogenic even in small amounts. Moreover, they often occur as mixtures of chlorinated alkenes, alkanes, and sometimes chloroform. Current technologies for remediating soils and/or groundwater contaminated with chlorinated solvents are limited by their high costs or the fact that they often do not destroy the contaminating chemicals. Biological oxidation of chlorinated solvents, however, results in the complete destruction of the contaminant, and degradative microorganisms can be used to destroy contaminants spread over large areas or trapped in aquifers. Currently available biological catalysts for destroying chlorinated solvents are somewhat limited by their narrow substrate ranges, the instability of the degradative enzymes, or inherent properties such as slow growth or sensitivities to co-contaminants which make their application commercially impractical. During this project Envirogen, Inc. proposes to use modern molecular biological techniques to develop biocatalysts which have improved enzyme substrate specificity's and stability while processing characteristics such as rapid growth and robustness which make their application commercially feasible.
