| 摘要: | 隨著人類的活動及科技的發展，為我們帶來工業的發展，但是石油、石化工業所生產的苯環化合物，卻造成了環境的污染。苯環類化合物之所以難分解是由於其具有穩定的苯環結構，因此會一直長期存在環境中，目前已知環境中許多細菌具有分解苯環類化合物的能力，其分解途徑與?的特性已被研究；本研究則以一本土性之苯環化合物分解菌(Pseudomonas putida SH1)為材料，此菌可以?、酚、鄰甲基苯酚、間甲基苯酚、對甲基苯酚、菲、芘、苯並芘為單一碳源生長，尤其以?或酚為單一碳源時生長狀況最好。當我們以不同的苯環化合物為唯一碳源培養P. putida SH1，已純化出四種苯環切割?(aromatic ring-cleavage dioxygenases)，其皆具鄰苯二酚加氧?之活性。在生化特性及NH2端胺基酸序列上皆顯示四個為不同之?，由?所誘發生成之catechol 2,3-dioxygenase (簡稱C23O)命名為C23Onap(SH1)，由酚誘發的有兩個分別為C23OpheI(SH1)及C23OpheII(SH1)，而以鄰甲基苯酚為唯一碳源生長時P. putida SH1可生成另一個C23O，命名為C23Oo-cre(SH1)。因之我們以其他已知之C23Os基因核?酸序列具高相似度的片段設計成引子，以聚合?連鎖反應選殖P. putida SH1中不同C23Os的基因，所放大的片段再經由pUC18-T載體接合，經由轉型至E. coli JM109並以C23O活性篩選和雜交兩種方法進行篩選SH1菌株中之C23O基因，所得兩個具有活性之片段與本研究室之前所選殖到的相同，故尚未發現其他C23O基因。 本論文另一個主題為利用苯環化合物分解菌中雙加氧?在基因上的相似性設計了幾組引子以偵測已知分解菌如可以分解?的P. putida G7及P. putida NCIB 9816-4，具分解甲苯之P. putida F1、P. putida mt-2及P. mendocina KR1，另外尚有能分解聯苯之P. putida LB400、能分解PAH之Sphingomonas B1及本實驗室篩出之菌株。PCR所用引子的設計為可偵測aromatic ring-hydroxylationg dioxygenase的ISP-F/ISP-R，是以此?中之終端加氧分子即iron-sulfur protein中a次單位的與輔因子( [2Fe-2S] Rieske center)結合的保守序列為主要偵測的位置；C23O-1F/C23O-1R、C23O-2F/C23O-2R與C23O-2’F/C23O-2R則是用在偵測間位苯環切割?的存在與否；另外還有針對?雙加氧?所設計的NahAc-F/NahAc-R及為偵測甲苯雙加氧酵?所設計的TodC1-F/TodC1-R用意在區別單和雙環苯環化合物之分解菌。在實驗中發現引子C23O-1F/C23O-1R的結果正確性很高，但是精確度不足；反之引子C23O-2F/C23O-2R及C23O-2’F/C23O-2R即是精確度很高但正確性不足，對於引子C23O-1F/C23O-1R的缺點，可以探針C23O-1經南方雜合加以相互印證；另外在偵測苯環化合物加氧?的實驗中，所設計的引子ISP-F/ISP-R的專一性很好，但是卻無法偵測到聯苯分解菌中的aromatic ring-hydroxylationg dioxygenase；專為?雙加氧?所設計的引子NahAc-F/NahAc-R可以成功的區分出?雙加氧?與單環的加氧?，但是卻無法偵測到同為?分解菌之P. putida SH1中之?雙加氧?，再經由探針NDO所進行南方雜合之結果發現P. putida SH1確實存在著一個NahAc-F/NahAc-R無法偵測到之?雙加氧?。以上引子並應用於未知代謝途徑之Triton X-100分解菌及6個經石油污染之土壤樣品中是否含有上述之?的存在，以初步鑑定出含有上述基因之存在，以供進一步快速偵測和研究之參考。 Aromatic compounds produced industrially from petrochemical industry were widespread and were important pollutants in the environment. They are classified into two groups, ring-hydroxylation dioxygenase and aromatic ring-cleavage dioxygenase. Pseudomonas putida SH1 was isolated from a contaminated soil in Taiwan. It was shown that this strain was capable to use naphthalene, phenol, o-cresol, m-cresol, p-cresol, pyrene, and phenanthrene as its sole source of carbon to grow. In the degradation of aromatic compounds by aerobic bacteria, two important steps are involved with oxygenase by the catalytic reaction of dioxygenase. From our previous study, four biochemically district catechol 2,3-dioxygenases (C23Os), ring-cleavage dioxygenases, had been purified from P. putida SH1. Polymerase chain reaction primers derived from the conserved sequence of three known C23O genes we used for the cloning of C23Os from P. putida SH1. Two 1.0 kb fragments showing C23O activity have been cloned in pUC18 but are the nucleotide sequence of these two insects showed extract the same to our previous cloned gene. Further screening of other insects need to be performed. The second part of this study was designed to detect the aromatic ring-hydroxylating dioxygenase and aromatic ring-cleavage dioxygenase genes in bacteria by PCR. The PCR method for the detection of naphthalene dioxygenase, toluene dioxygenase and catechol 2,3-dioxygenase were performed. The first two dioxygenases are two archetypical ring-hydroxylation dioxygenase. The catechol 2,3-dioxygenase is involved in the cleavage of aromatic ring and shows high identify among C23O genes from many bacteria. Strains test in this study included naphthalene-degrading bacteria, P. putida G7 and P. putida NCIB 9816-4; toluene-degrading bacteria, P. putida F1, P. putida mt-2 and P. mendocina KR1; biphenyl-degrading bacteria, P. putida LB400; polycyclic aromatic hydrocarbons-degrading bacteria, Sphingomonas B1, and bacterial strains isolated by our laboratory. In addition to petrochemical hydrocarbons-degrading bacteria, bacteria degrading Triton X-100 were also included. From our preliminary study, primers C23O-1F/C23O-1R are not very specificity, but if combined with Southern hybridization by probe C23O can accurate to detect aromatic ring-cleavage dioxygenase genes in bacteria. Primers C23O-2F/C23O-2R and C23O-2'F/C23O-2R have specificity, but they have low accuracy for detect aromatic ring-cleavage dioxygenase genes in bacteria. Primers NahAc-F/NahAc-R are very accurate for detect naphthalene dioxygenase genes, one exception is the naphthalene-degrading P. putida SH1. When we used Southern hybridization by probe NDO, which showed low sequence similarity with P. putida G7. Primers ISP-F/ISP-R are very specificity for detect aromatic ring-hydroxylating dioxygenase. Combined with Southern hybridization, we can get the powerful tool to detect these genes not only using conserved primer sequence but also using full sequence similarity. This detection method will be applied to pretiminary screen of the key degradation genes in unknown isolates as well as for detecting aromatic compound-degrading bacteria in the environment. |
