Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36197
標題: 選殖Arthrobacter simplex之3-酮類固醇-9α-羥基化酵素
Cloning the 3-ketosteroid 9-α-hydroxylase gene from Arthrobacter simplex
作者: 張哲銘
Chang, Che-Ming
關鍵字: 3-ketosteroid 9-α-hydroxylase;3-酮類固醇-9α-羥基化酵素;Arthrobacter simplex
出版社: 生物科技學研究所
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摘要: 
類固醇是一群具有重要生理活性的化合物,也是全世界各大藥廠研發的重點,因此類固醇藥物的經濟價值相當可觀。早期合成類固醇藥物以化學方法為主,不但合成效益不高且其廢棄物也會造成環境問題,便以微生物轉換方法來取代部分的類固醇合成步驟。3-酮類固醇9-α-羥基化酵素是降解類固醇環系結構的重要酵素,目前在Mycobacterium、Arthrobacter、Nocardia及Rhodococcus都發現有3-酮類固醇-9α-羥基化酵素的活性。在工業生產上也利用3-酮類固醇-Δ1,2-脫氫酵素或3-酮類固醇9-α-羥基化酵素缺陷的Mycobacterium,累積9-α-羥基雄烯二酮或雄二烯二酮作為類固醇藥物生產的前驅物,得以用來合成高經濟價值的類固醇藥物。由於Arthrobacter simplex 對膽固醇有較高的耐受性,是工業發酵培養上極具潛力的菌種,而且A. simplex的3-酮類固醇-9α-羥基化酵素更詳細的相關研究也尚未被發表,便以此作為研究材料。利用文獻發表過的3-酮類固醇-9α-羥基化酵素及相關氧化酵素的胺基酸序列,進行多序列比對尋找各序列的保留區域,接著由保留區域的胺基酸序列設計退化性引子進行聚合酶連鎖反應,試圖得到A. simplex 的3-酮類固醇9-α-羥基化酵素。將得到的基因片段定序之後並選殖出來,接著利用反向-聚合酶連鎖反應及DNA步行的方式找出保留區域上、下游的序列,得到完整的酵素序列。結果得知A. simplex的3-酮類固醇-9α-羥基化酵素是由kshA(末端氧化酶)及kshB(還原酶)兩個組元所組成,其中kshA的部分與Rhodococcus一樣具有兩個同功形態,kshA(S1A2)及kshA(S3A2),然而在kshB的部分並沒有發現其他同功形態。進一步利用workbench針對kshA(S1A2)與kshA(S3A2)的完整DNA序列以及胺基酸序列來進行整體排比,兩條序列在DNA層次有著70.5%的相似度;在胺基酸層次,則有58.7%的相似程度。然而兩個同功形態在3-酮類固醇-9α-羥基化酵素中扮演的角色以及活性能力,都得進一步進行活性分析才得以判斷。確認3-酮類固醇9-α-羥基化酵素完整的核酸序列之後,便可以利用限制酵素切割成不完整的3-酮類固醇9-α-羥基化酵素再經由基因重組方式送入A. Simplex形成3-酮類固醇9-α-羥基化酵素的缺陷株,接著便可分析是否有較佳的膽固醇中間代謝物累積的能力,以適用於工業發酵生產之上。

Steroids, which are focused by many pharmaceutical companies, possess considerable bioactivities and huge economic value. In early period, chemical synthesis of steroid compounds was the major process, however the synthetic efficiency was low and the waste disposal caused a serious environmental problem. Therefore, the steroid synthesis coupled the chemical and biological approaches. The 3-ketosteroid 9-α-hydroxylase (ksh) is the key enzyme in the steroid degradation and has been discovered in various actinobacterial genera, e.g., Arthrobacter, Nocardia, Mycobacterium, and Rhodococcus. The steroid industry utilize the 3-ketosteroid- Δ1,2-dehydrogenase or ksh enzymes mutant Mycobacterium to accumulate the steroid pharmaceutical precursors, 9α-hydroxyandrost-4-ene-3,17-dione or 1,4-androstadiene- 3,17-dione, to synthesize high value steroid. A. simplex is a potential strain in industrial fermentation on the basis of higher tolerance to cholesterol. Besides, more detailed research of A. simplex has not yet been published. Because of these reasons, we choose A. simplex as the material in this study. At first, we blasted some ksh enzymes and related oxygenase sequences published and search the conserve regions to design degenerated primer. After PCR amplification, the fragment were sequenced and cloned. In order to obtain the entire sequence, we performed inverse PCR and DNA walking, the result of which showed that ksh enzymes in A. simplex consisted of two components, kshA (terminal oxygenase) and kshB (reductase). Moreover, there are two kshA isoforms, kshA(S1A2) and kshA(S3A2). We next used global alignment to align these two isoforms in workbench website. It showed 70.5% identity in DNA and 58.7% in amino acid level between these two isoforms. We shall assay the enzyme activity of these two isoforms to identify their role and the activity in steroid degradation. Finally, we shall use restriction enzyme to generate truncated 3-ketosteroid 9-α-hydroxylase, and perform genetic recombination to generate mutant A. simplex. In the future, we will assay whether the 3-ketosteroid 9-α-hydroxylase knockout strain has higher accumulation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione.
URI: http://hdl.handle.net/11455/36197
其他識別: U0005-0202201017505300
Appears in Collections:生物科技學研究所

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