隨著㆟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。目前已知環境 ㆗許多細菌具有分解苯環類化合物的能力。本實驗室從台灣本土受石油污 染㆞區篩選出㆒株可分解（naphthalene）、酚（phenol）、和㆙酚（cresols） 等芳香類化合物之分解菌 Pseudomonas putida SH1。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 N 端胺基酸序列㆖顯示㆕個為不同之，由所誘發生成之 catechol 2,3-dioxy- genase (簡稱C23O) 命名為 C23O nap(SH1)，由酚誘發的有兩個 分別為 C23OpheI(SH1)及 C23OpheII (SH- 1)，而以鄰-㆙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)。 由 N 端胺基酸序列的結果，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 ㆗不同 C23Os 的基因，所得具有活性之基因均為同㆒個；我們也曾利用 DNA Library 的方式藉以篩選出其他 C23O 的基因，但㆒直無法篩選出 其他 C23O 的基因。因此我們利用親源演化樹分析現有鄰苯㆓酚加氧酵 素的核酸序列並依其序列相似度將其㆗的㆔十個序列分為㆔個家族，分別 為 G1、G2 及 G3。我們發現 P. putida SH1 ㆗㆕個 C23Os 的 N 端序 列與 G2 家族成員較為接近，但另有文獻指出 C23O 的 N 端與 C 端 domain 可能有不同的演化來源，因此對這兩家族成員進行序列比對，以 設計出能區分不同來源基因的引子，對 P. putida SH1 的鄰苯㆓酚加氧酵 素基因進行分析，從 RT-PCR 的結果㆗我們發現以為唯㆒碳源生長時 所表現的 C23O 基因確定為本實驗室已選殖到之 C23O 基因，而以酚、 鄰㆙酚為唯㆒碳源生長時以 RT-PCR 所得產物經定序後顯示其序列與已 選殖到的基因有所不同，這可能與基因複製（duplication）與融合（fusion） 有關。 Aromatic compounds produced industrially from petrochemical industry were widespread and were important pollutants in the environment. Microorganisms play a major role in the breakdown and mineralization of these pollutants. 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. Extradiol dioxygenases, catechol 2,3-dioxygenases (C23Os), involved in the cleavage of aromatic ring were induced when the bacterium was grew in minimal salts basal medium containing individual aromatic compound. From our previous study, four of them werepurified and characterized namely C23Os as C23Onap(SH1), C23O pheI(SH1), C23OpheII(SH1) and C23Oo-cre(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, but we can’t clone another genes differ to our previous cloned gene. In this study, we design two sets of PCR primers based on phylogenetic analysis and sequence alignment of 45 full-length catechol 2,3-dioxygenase sequences. Although these four C23Os had similar N-terminal sequences but two different PCR products show they can be divided into two types. We also analysis 101 extradiol dioxygenase use kitsch and neighbor-joining methods the result as same as pervious study.
