P450 SYSTEMS IN MICROBIAL BIODEGRADATION

该项目的长期目标是获得新的分子 协作中使用的微生物代谢的遗传理解 研究开发改进的生物处理技术。 目标化合物是对环境不利的多芳香族化合物 碳氢化合物和 N-杂环化合物 (PAH/NHA) 以及磺化偶氮染料。 重点是通过细胞色素 P450 进行有氧代谢 第一类是单氧化，通过对氧不敏感的偶氮进行 偶氮染料的键断裂。 真菌和/或细菌微生物 已知可以执行这些解毒步骤，包括 一些在该计划的其他项目中是孤立的。 P450基因 将从处理重要单加氧酶的微生物中分离出来 通过序列修饰和 在大肠杆菌中的表达。 相对较少的微生物 P450 具有遗传特征，但现有证据表明 存在大量高度不同的异构体，仅以 每种微生物有几种异构体。 为了分离 P450 基因，我们提出了 可能是针对最保守的小片段的通用方法 这个高度序列趋异的基因超家族的区域。 具体目标 1 和 2：我使用丝状真菌测试了两种相关的 PCR 策略 分别为秀丽隐杆线虫和土壤分枝杆菌菌株 PAH/N-杂环化合物代谢的P450基因的分离；具体 目标 3：基于纯化的 P450 酶分离 P450 基因 Cunninghamella sp，作为目标 1 的替代方案：具体目标 4：分离 并表征需氧革兰氏菌的氧不敏感偶氮还原酶基因 负极棒C7。 预先纯化酶以开发基因探针； 具体目标 5：使用特定基因探针准备和测试通路 生物降解系统中微生物群落的合作研究。

The long-term objective of this project is to acquire new molecular genetic understanding of microbial metabolism of use in collaborative research on the development of improved biotreatment technologies. Targeted compounds are environmentally recalcitrant polyaromatic hydrocarbons and N-heterocyclics (PAH/ NHA), and sulfonated azo dyes. The emphasis is upon aerobic metabolism via cytochrome P450 monooxygenation for the first category, and via oxygen insensitive azo bond cleavage of the azo dyes. Fungal and/or bacterial microorganisms known to carry out these detoxication steps are available, including several that were isolated in other projects of this program. P450 genes will be isolated from microbes processing important monooxygenases and characterized for their enzyme products, by sequence modification and expression in Escherichia coli. Relatively few microbial P450s have been genetically characterized but the available evidence indicates the existence of a large number of highly divergent isomers, present as only a few isomers per microbe. For isolation of P450 genes we propose a possibly general method that targets small segments in the most conserved region of this highly sequence divergent gene superfamily. Specific Aims 1 and 2: I test two related PCR strategies using the filamentous fungus Cunninghamella elegans and soil mycobacterium strains respectively, for the isolation of P450 genes of PAH/N-heterocyclics metabolism; Specific Aim 3: Isolate P450 genes based upon P450 enzymes purified from Cunninghamella sp, as an alternative to Aim 1: Specific Aim 4: Isolate and characterize oxygen insensitive azo reductase genes from aerobic Gram negative rod C7. Previously purified enzyme to develop a gene probe; Specific Aim 5: Prepare and test pathway using specific gene probes for collaborative studies of microbial communities in biodegradation systems.