SBIR Phase II: Molecular Engineering of a Biocatalyst for the Destruction of Chlorinated Solvents

*** 9623832 Steffan这个小型企业创新研究二期项目旨在生产具有成本效益的生物催化剂，用于氯化溶剂污染环境的生物修复。甲苯-4-单加氧酶（T4MO）是一种来自mendocina假单胞菌KRl的多组分酶，能够氧化氯化溶剂，直接导致其生物修复。在第一阶段，确定了几个控制T4MO反应性的氨基酸序列区域。然后使用体外诱变技术（位点定向或随机引物聚合酶链反应）改变这些区域的氨基酸序列。这一努力产生了新的、具有催化活性的T4MO突变异构体。这些新异构体中最有希望的一种对卤化碳氢化合物（如三氯乙烯（TCE）和氯仿）的氧化速率增加，而其他几种异构体相对于野生型酶表现出底物特异性的变化。这些结果证明了该项目通过基因工程提高T4MO酶的催化多功能性策略的合理性。这两种突变异构体在商业应用中都是有价值的。拟议的二期项目将详细评估这些有前景的突变体的商业潜力，包括进一步表征氧化速率、底物特异性范围、产品分布、催化剂稳定性、生物反应器放大参数以及这些新型工程生物催化剂的其他相关结构和功能特性。在这个项目中开发的生物催化剂将在商业上适用于在政府和私人场所修复许多国家受污染的含水层和土壤。最初的应用将在密闭气相生物反应器中，未来的应用将包括原位处理。＊＊＊

*** 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. ***