Molecular Engineering of a Biocatalyst for the Destruction of Chlorinated Solvents

这个小企业创新研究第一阶段项目涉及使用分子生物学技术来改变生物催化剂的底物特异性和活性，以破坏各种顽固的氯化溶剂。mendocina假单胞菌（Pseudomonas mendocina）克隆的甲苯单加氧酶基因KR1的推定活性位点将通过位点定向诱变进行改变，使其更接近于广泛底物特异性甲烷单加氧酶的活性位点。所得到的催化剂将被测试其降解各种氯化溶剂的能力，到目前为止还没有被野生型生物降解。在假定的酶的氧结合区改变的影响也将被检查。氯化溶剂是美国地下水和土壤中分布最广、含量最多的污染物，其中许多即使是少量也会直接致癌。此外，它们通常是氯化烯烃、烷烃，有时是氯仿的混合物。目前用于修复被氯化溶剂污染的土壤和/或地下水的技术由于成本高或往往不能破坏污染化学品而受到限制。然而，氯化溶剂的生物氧化导致污染物的完全破坏，降解微生物可用于破坏大面积扩散或被困在含水层中的污染物。目前可用的用于破坏氯化溶剂的生物催化剂在一定程度上受到底物范围狭窄、降解酶的不稳定性或固有特性（如生长缓慢或对共污染物敏感）的限制，这使得它们在商业上的应用不切实际。在这个项目中，Envirogen公司建议使用现代分子生物学技术来开发生物催化剂，这些生物催化剂具有提高酶底物特异性和稳定性的同时，还具有快速生长和坚固性等特性，使其在商业上的应用具有可行性。

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.