Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22706
標題: 應用紫色不含硫光合菌表現假單胞菌細胞色素P450cam於生物降解五氯乙烷之研究
Biodegradation of Pentachloroethane by expressing the Pseudomonas putida Cytochrome P450cam in the purple non-sulfur phototrophic bacteria
作者: 林佳靜
Lin, Chia-Ching
關鍵字: halorespiration
鹵化呼吸作用
Rhodopseudomonas palustris CGA009
cytochrome P450cam
Pseudomonas putida PpG1
紫色不含硫光合菌CGA009
細胞色素P450cam
出版社: 生命科學系所
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摘要: 環境中的污染源像是三氯乙烯和四氯乙烯這些含氯碳氫化合物可在厭氧的環境之下,經由微生物的鹵化呼吸作用將其進行還原性脫氯使其變成含氯較低的含氯碳氫化合物,或者無毒害性的物質。鹵化呼吸作用主要是由電子提供者、電子攜帶者以及電子接受者所組成。大多數的微生物須利用氫氣當作電子提供者行使鹵化呼吸作用,因此與可以利用提供氫氣的產氫微生物以共培養的方式解決這些含氯碳氫化合物。 本研究是以構築Pseudomonas putida PpG1質體上的細胞色素P450cam卡匣基因(camC、camA、camB)當作電子攜帶者轉殖於可光合產氫的紫色不含硫光合菌裡表現,其細胞在厭氧狀態時五氯乙烷此含氯碳氫化合物會在原本樟腦結合camC之活化中心位置進行還原性脫氯作用。因此本實驗以五氯乙烷為主要研究目標,研究探討細胞色素P450cam在紫色不含硫光合菌CGA009裡對於五氯乙烷表現的情形。 在含有構築選殖載體 pMG105、pMG-camC和pMG-camCAB之紫色不含硫光合菌CGA009的RNA,以RT-PCR方式偵測是否所構築之細胞色素P450的選殖載體能否在紫色不含硫光合菌CGA009裡表現 。 以厭氧方式的培養含有不同的構築選殖載體pMG105、pMG-camC和pMG-camCAB之紫色不含硫光合菌CGA009對於五氯乙烷降解分析之比較上,含有pMG-camCAB選殖載體的紫色不含硫光合菌CGA009,其在降解的速率或者產氫的能力比pMG105、pMG-camC比較上要來得有優異的表現。 而以苯環類的含氯化何物3-單氯苯甲酸作為碳源,發現轉殖株在生長測試方面並無明顯的差異性存在,顯示在本實驗裡可能不具有可將3-單氯苯甲酸以及苯甲酸分解代謝利用的現象。
Environmental contaminants, such as anthropogenic chlorinated compounds tri-chloroethylene (TCE) and tetrachloroethylene (PCE), can be reductively dehalogenated to less chlorinated ethenes or innocuous compounds by micro-organisms through halorespiration under anaerobic condition. The halorespiration is composed of electron donor, electron carrier, and electron acceptor. Most of these microorganisms can use hydrogen as electron donor to proceed reductive dehalogenation. The decomposition of chlorinated compound as the major targets for bioremediation with hydrogen-producing microorganisms by using co-culture. Here we construct the genes enconding cytochrome P450cam gene (camC, camA, and camB) expressed in a photosynthetic hydrogen-producing microorganisms Rhodo-pseudomonas palustris CGA009. Under anoxic condition, pentachloroethane (PCA) that bind at the camphor-binding site could be reduced by the camC of cytochrome P450cam gene. We use pentachloroethane as a model substrate for characterizing genetically engineered bacteria containing theses enzyme systems. The Rhodopseudomonas palustris CGA009 express gene including pMG105, pMG-camC, and pMG-camCAB were analyzed by using RT-PCR to monitor RNA expression. In order to analysis the biodegradation of pentachloroethane, the engineered strain carried the pMG-camCAB plasmid exhibited the predominant rate of the PCA degradation and production of hydrogen rather than pMG105 and pMG105-camC strains. Two strains of pMG105 and pMG-camCAB absence of the ability to grow on 3-Chlorobenzoate and 3-Chlorobenzoate plus Benzoate as carbon source. Probably no chlorinated benzoate or benzoate were degraded by Rhodopseudomonas palustris CGA009 two strains.
URI: http://hdl.handle.net/11455/22706
其他識別: U0005-1908200821502200
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1908200821502200
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