P450 SYSTEMS IN MICROBIAL BIODEGRADATION

该项目的长期目标是获得新的分子 微生物代谢的遗传学理解， 研究改进生物处理技术的发展。 目标化合物是对环境无害的多环芳烃 碳氢化合物和N-杂环化合物（PAH/ NHA）和磺化偶氮染料。 重点是有氧代谢通过细胞色素P450 第一类为单加氧，第二类为氧不敏感偶氮 偶氮染料的键断裂。 真菌和/或细菌微生物 已知进行这些解毒步骤的方法是可获得的，包括 有几个在这个项目的其他项目中被孤立。 P450基因 将从处理重要单加氧酶的微生物中分离， 通过序列修饰表征其酶产物， 在大肠杆菌中表达。 相对较少的微生物P450已经 基因特征，但现有证据表明， 存在大量高度不同的异构体，仅以 每个微生物有几个异构体。 为了分离P450基因，我们提出了一种 可能的通用方法，目标是最保守的小片段， 这个高度序列差异的基因超家族的区域。 具体目标 图1和图2：我使用丝状真菌测试了两种相关的PCR策略 雅致小克银汉霉和土壤分枝杆菌菌株， PAH/N-杂环类代谢相关P450基因的分离;特异性 目的3：基于从大肠杆菌中纯化的P450酶分离P450基因。 小克银汉霉属，作为目标1的替代：具体目标4：分离株 并表征来自好氧革兰氏菌的氧不敏感偶氮还原酶基因 负极棒C7。 先前纯化的酶，以开发基因探针; 具体目标5：使用特异性基因探针制备和测试通路， 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.