Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36197
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dc.contributor劉文雄zh_TW
dc.contributorWen-Hsiung Liuen_US
dc.contributor楊昭順zh_TW
dc.contributorChao-Hsun Yangen_US
dc.contributor.advisor孟孟孝zh_TW
dc.contributor.advisorMenghsiao Mengen_US
dc.contributor.author張哲銘zh_TW
dc.contributor.authorChang, Che-Mingen_US
dc.contributor.other中興大學zh_TW
dc.date2011zh_TW
dc.date.accessioned2014-06-06T07:54:10Z-
dc.date.available2014-06-06T07:54:10Z-
dc.identifierU0005-0202201017505300zh_TW
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Oxygenation of steroids by Mucorales fungi. U.S. Patent 2602769. Ochman, H., A. Gerber and D. Hartl (1988). "Genetic application of an inverse polymerase chain reaction. ." Genetics 120(621-623). Perry, L. L. and G. J. Zylstra (2007). "Cloning of a gene cluster involved in the catabolism of p-nitrophenol by Arthrobacter sp. strain JS443 and characterization of the p-nitrophenol monooxygenase." J. Bacteriol. 189(21): 7563-7572. Petrusma, M., L. Dijkhuizen and R. van der Geize (2009). "Rhodococcus rhodochrous DSM 43269 3-ketosteroid 9alpha-hydroxylase, a two-component iron-sulfur- containing monooxygenase with subtle steroid substrate specificity." Appl. Environ. Microbiol. 75(16): 5300-5307. Rosenthal, A. and D. S. Jones (1990). "Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction." Nucleic Acids Res. 18(10): 3095-3096. Schneider, D. and C. L. Schmidt (2005). "Multiple Rieske proteins in prokaryotes: where and why?" Biochim. Biophys. Acta 1710(1): 1-12. Siebert, P. D., A. Chenchik, D. E. Kellogg, K. A. Lukyanov and S. A. Lukyanov (1995). "An improved PCR method for walking in uncloned genomic DNA." Nucleic Acids Res. 23(6): 1087-1088. Stadtman, T. C., A. Cherkes and C. B. Anfinsen (1953). "Studies on the microbiological degradation of cholesterol." J. Biol. Chem. 206(2): 511-523. Strijewski, A. (1982). "The steroid-9 alpha-hydroxylation system from Nocardia species." Eur. J. Biochem.128(1): 125-135. Szentirrnai, A. (1990). "Microbial physiology of sidechain degradation of sterols " J. Ind. Microbiol. 6: 101-116. Van der Geize, R., G. I. Hessels, M. Nienhuis-Kuiper and L. Dijkhuizen (2008). "Characterization of a second Rhodococcus erythropolis SQ1 3-ketosteroid 9alpha-hydroxylase activity comprising a terminal oxygenase homologue, KshA2, active with oxygenase-reductase component KshB." Appl. Environ. Microbiol. 74(23): 7197-7203. Van der Geize, R., G. I. Hessels, R. van Gerwen, P. van der Meijden and L. Dijkhuizen (2002). "Molecular and functional characterization of kshA and kshB, encoding two components of 3-ketosteroid 9alpha-hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1." Mol. Microbiol. 45(4): 1007-1018. Van der Geize, R., K. Yam, T. Heuser, M. H. Wilbrink, H. Hara, M. C. Anderton, E. Sim, L. Dijkhuizen, J. E. Davies, W. W. Mohn and L. D. Eltis (2007). "A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages." Proc. Natl. Acad. Sci. USA. 104(6): 1947-1952. Wang, F. Q., B. Li, W. Wang, C. G. Zhang and D. Z. Wei (2007). "Biotransformation of diosgenin to nuatigenin-type steroid by a newly isolated strain, Streptomyces virginiae IBL-14." Appl. Microbiol. Biotechnol. 77(4): 771-777. Yanofsky, M. F., H. Ma, J. L. Bowman, G. N. Drews, K. A. Feldmann and E. M. Meyerowitz (1990). "The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors." Nature 346(6279): 35-39.zh_TW
dc.identifier.urihttp://hdl.handle.net/11455/36197-
dc.description.abstract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。zh_TW
dc.description.abstractSteroids, 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.en_US
dc.description.tableofcontents目次 第一章、 緒言 1 一、 類固醇 1 二、 類固醇藥物生產 .3 三、 微生物轉換生產類固醇藥物 5 四、 3-酮類固醇9α-羥基化酵素的特性 8 五、 關節桿菌簡介與相關育種研究 13 六、 研究動機與目的 15 第二章、 材料與方法 16 第一節、 Arthrobacter simplex 16 一、 菌種 16 二、 培養基 16 三、 生長曲線製作 16 四、 菌種保存 18 五、 基因組DNA萃取 18 (一) 各式溶液、緩衝液配製 18 (二) 基因組DNA萃取方法 19 第二節、 聚合酶連鎖反應 20 一、 退化性引子設計 20 第三節、 TA cloning. 22 一、 緒論 22 二、 大腸桿菌轉型作用 22 (一) 大腸桿菌菌株 22 (二) 各式培養基、溶液、緩衝液配製 22 (三) 大腸桿菌勝任細胞的製備 23 (四) 轉型作用與篩選與篩選 25 (五) 大腸桿菌質體DNA之抽取 25 (六) 質體保存與核酸定序 26 第四節、 選殖側翼DNA片段的方法 27 (一) 緒言 27 (二) 反向-聚合酶連鎖反應 28 (三) DNA步行 31 第三章、 結果 36 一、 保留性區域序列分析 36 二、 側翼序列分析 41 三、 3-酮類固醇-9α-羥基化酵素的分析 .43 (一) kshA(S1A2) 43 (二) kshA(S3A2) 48 (三) kshA(S1A2)與kshA(S3A2)的比較 53 (四) kshB 59 第四章、 討論 63 第五章、 參考文獻 66 表目次 表一、 目前以運用於工業規模生產之環系酵素缺陷株 7 表二、 ROs的系統分類 12 表三、 退化性引子的序列 21 表四、 反向-聚合酶連鎖反應所使用的引子 30 表五、 DNA步行所反應所使用的引子 32 表六、 DNA步行套組所附引子序列 34 表七、 DNA步行的反應條件 35 表八、 kshA(S1A2)比對結果 47 表九、 kshA(S3A2)比對結果 52 表十、 kshB比對結果 62 圖目次 圖1.1 類固醇分子的結構以及其命名編號 .2 圖1.2 類固醇生產製程總覽圖 4 圖1.3 微生物完全降解膽固醇的反應路徑 6 圖1.4 3-酮類固醇-9α-羥基化酵素的特性 11 圖1.5 Arthrobacter simplex之育種過程 14 圖2.1 A. simplex的生長曲線 17 圖2.2 TA cloning所用的載體 24 圖2.3 反向-聚合酶連鎖反應原理 29 圖2.4 DNA步行反應原理總覽 33 圖3.1-1 3-酮類固醇-9α-羥基化酵素相關酵素序列進行多序列比對 37 圖3.1-2 聚合酶連鎖反應結果 38 圖3.1-3 BLAST所得到的結果 39 圖3.1-4 F1-60-1(S1A2)與F1-60-1(S3A2)部分序列比對 .40 圖3.2-1 F1-60-1(S1A2)片段進行反向-聚合酶連鎖反應 42 圖3.3-1 S1A2的定序策略 44 圖3.3-2 S1A2的完整序列與其上下游序列 45 圖3.3-3 S3A2的定序策略 49 圖3.3-4 S3A2的完整序列與其上下游序列 50 圖3.3-5 S1A2與S3A2於DNA層次的整體排比 54 圖3.3-6 S1A2與S3A2於胺基酸層次的整體排比 57 圖3.3-7 kshB的定序策略 60 圖3.3-8 kshB的部分序列與其下游序列 61zh_TW
dc.language.isoen_USzh_TW
dc.publisher生物科技學研究所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0202201017505300en_US
dc.subject3-ketosteroid 9-α-hydroxylaseen_US
dc.subject3-酮類固醇-9α-羥基化酵素zh_TW
dc.subjectArthrobacter simplexen_US
dc.title選殖Arthrobacter simplex之3-酮類固醇-9α-羥基化酵素zh_TW
dc.titleCloning the 3-ketosteroid 9-α-hydroxylase gene from Arthrobacter simplexen_US
dc.typeThesis and Dissertationzh_TW
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