P450 SYSTEMS IN MICROBIAL BIODEGRADATION

这个项目的长期目标是获得新的分子 对微生物代谢的遗传学认识在协作中的应用 研究开发改良的生物处理技术。 目标化合物是环境顽固的多芳香族 碳氢化合物和N-杂环化合物(PAH/NHA)和磺化偶氮染料。 重点是细胞色素P450的有氧代谢。 第一类为单氧化，并通过氧不敏感的偶氮 偶氮染料的键断裂。真菌和/或细菌微生物 已知可执行这些戒毒步骤，包括 在这个项目的其他项目中被隔离的几个。P450基因 将从处理重要单加氧酶的微生物中分离出来 以其酶产物为特征，通过序列修改和 在大肠杆菌中表达。微生物P450相对较少 具有基因特征，但现有证据表明 存在大量高度发散的异构体，仅以 每个微生物只有几个异构体。为了分离P450基因，我们提出了一种 可能是以最保守的小片段为目标的通用方法 这个高度序列分化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 克氏杆菌和土壤分枝杆菌分别用于 多环芳烃/N-杂环化合物代谢P450基因的分离 目的3：分离基于纯化的P450酶的P450基因 Cunninghamella sp，作为目标1的替代：特定目标4：分离 并从需氧革兰氏菌中鉴定氧不敏感偶氮还原酶基因 负极棒C7。预先纯化的酶，以开发基因探针； 特定目标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.