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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李平篤 | |
| dc.contributor.author | Che-Jen Hsiao | en |
| dc.contributor.author | 蕭哲仁 | zh_TW |
| dc.date.accessioned | 2021-06-13T15:18:16Z | - |
| dc.date.available | 2008-07-26 | |
| dc.date.copyright | 2008-07-26 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-07-25 | |
| dc.identifier.citation | 1. Jensen, W. B. J., The Origin of the Names Malic, Maleic, and Malonic Acid. 2007; Vol. 84, p 924.
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J Biosci Bioeng 2003, 95, (6), 562-6. 莊榮輝 (1985) 博士論文，國立臺灣大學農業化學研究所 謝陸盛 (2003) 碩士論文，國立臺灣大學農業化學研究所 謝陸盛 (2005) 博士論文初稿，國立臺灣大學微生物與生化學研究所 莊緒怡 (2006) 碩士論文，國立臺灣大學微生物與生化學研究所 盧厚任 (2007) 碩士論文，國立臺灣大學微生物與生化學研究所 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37041 | - |
| dc.description.abstract | 蘋果酸去氫酶 (malate dehydrogenase, MDH EC 1.1.1.37) 在生物體內分佈非常廣泛，負責可逆性催化oxaloacetate 和malate間的轉換。高等植物中依照對不同輔酶的專一性、胞內位置和生理功能，而有不同的異構酶，其中利用NAD和位於乙醛酸循環體 (glyoxysome) 的gMDH是一種植物中尚未被完全研究的MDH。
本實驗室之前於綠竹cDNA庫篩選細胞分裂素氧化酶/去氫酶時，意外篩到了一可能是綠竹gMDH的基因，因而在此篇論文裡我們將對此基因進行選殖、原核表現和檢定。回推基因庫發現此gMDH不具任何內插子，而蛋白質序列分析則顯示和許多其他植物的gMDH有高相似性，其中和同為禾本科之稻米相似度更達96%。在N端接上Trx-His tag並透過大腸桿菌BL21 (DE3) 表現出約57 kD的重組蛋白質，也顯示了高度MDH活性，在在都確認了此一基因即綠竹乙醛酸循環體的MDH。 以重組蛋白質製備MDH多株抗體後，從綠竹筍中直接純化MDH。經緩衝液粗抽、硫酸銨分劃、親和層析法 (Blue Sepharose¬¬¬ CL-6B)、膠體過濾法 (Superose 12 HR 10/30) 得到的純化蛋白質，和重組蛋白質在動力學和最適反應pH上有相似的性質：對malate kcat/Km約150、對NAD+ 約2500，最適反應pH為9.5。但在最適反應溫度上，重組MDH偏好低溫環境而純化MDH卻偏好高溫。以膠體過濾法測得MDH原態分子量介於130~115 kD，次單元分子量由SDS-PAGE得約45 kD，推測應為同質二元體結構。 | zh_TW |
| dc.description.abstract | Malate dehydrogenase (MDH EC 1.1.1.37), which is ubiquitous in nature, catalyzes the interconversion of oxaloacetate and malate. Higher plants contain multiple forms of MDH that differ in co-enzyme specificity, subcellular localization and physiological function. Glyoxysomal NAD-dependent MDH (gMDH) is one class of MDH that has not been extensively characterized in plants, and also there’s no any research studying the relation between gMDH and cytokinin oxidase/dehydrogenase (CKX, EC 1.55.99.12). Unexpectedly, a putative Mdh cDNA was screened with the specific probe of CKX from the cDNA library of Bambusa oldhamii in our laboratory.
Here we present the cloning, characterization and prokaryotic expression of this putative Mdh. Sequence alignment shows that there’s a high homology between the deduced amino sequence of BogMDH and MDH protein in glyoxysome in Oryza sativa (96%). Nearly 57 kD fusion protein was expressed by IPTG induction in Escherichia coli BL21 (DE3), and an obvious MDH activity was detected in the protein. All these results suggest that BogMDH encodes a glyoxysomal MDH. Screened the genomic library indicated the Mdh has no intron. After preparing the polyclonal antibody by using fusion protein as the antigen, we partial purified MDH from bamboo through buffer extraction, ammonium sulfate precipitation, and Fast Protein Liquid Chromatography (FPLC). The recombinant MDH and the purified MDH both have similar characteristics in kinetics and the optimum pH. But the recombinant protein prefers a low temperature. In contrast, the purified protein favors a higher temperature. Using Superose 12 column, the molecular weight of native form bamboo MDH was estimated to be 130 ~ 115 kD, and the subunit form was about 45 kD by SDS-PAGE. It might be a homodimeric enzyme. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T15:18:16Z (GMT). No. of bitstreams: 1 ntu-97-R95b47212-1.pdf: 2383576 bytes, checksum: e0b88aae0cbd3810b4af24c3ea7c4a20 (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 縮寫表 IV
摘要 V Abstract VI 第一章 緒論 1 第一節 實驗緣起 1 第二節 蘋果酸 2 第三節 Malate參予的生理反應 3 第四節 Malate含量的調控與相關酵素研究 7 第五節 蘋果酸去氫酶 8 第六節 MDH基因選殖 9 第七節 環境對MDH的影響 11 第八節 酵母菌和阿拉伯芥MDH基因啟動子之研究 12 第九節 植物中MDH異構酶的研究 15 第十節 MDH蛋白質結構與作用機制 18 第十一節 MDH在演化上的相關研究 23 第十二節 MDH與CKX的相關研究 26 第二章 材料與方法 27 第一節 實驗材料、藥品與儀器 27 1.1 實驗材料 27 1.2 實驗藥品與儀器 28 第二節 一般實驗法 29 2.1 瓊脂糖膠體電泳 29 2.2 質體DNA的小量分離 31 2.3 PCR clean-up 31 2.4 電穿孔法competent cell之製備 32 2.5 氯化鈣法competent cell之製備 34 2.6 PEG法competent cell之製備 35 2.7 Malate dehydrogenase酵素活性測定 36 2.8 蛋白質定量法 38 2.9 蛋白質電泳檢定系統 40 2.0 蛋白質轉印法 46 2.a 免疫染色法 47 2.b DNA定序 50 第三節 Escherichia coli BL21 (DE3) 表現系統之建立 50 3.1 聚合酶鏈鎖反應 (PCR) 51 3.2 DNA之限制酶分析 55 3.3 接合反應 57 3.4 pET-32a(+) 重組載體之建構、轉形與檢定 58 第四節 E. coli Rosetta-gami B (DE3) 表現系統之建立 61 4.1 pET-32a(+) 重組載體之建構 62 4.2 pET-32a(+) 重組載體之轉形與檢定 63 第五節 綠竹基因組 (genomic DNA) 之篩選 65 5.1 綠竹基因組的抽取 65 5.2 T&A cloning vector之建構、轉形與檢定 66 第六節 綠竹malate dehydrogenase之大量表現並純化 68 6.1 E. coli BL21 (DE3) 轉型株表現型之檢定 68 6.2 綠竹malate dehydrogenase最適誘導時間點之探討 69 6.3 綠竹malate dehydrogenase之大量表現 71 6.4 經表現之綠竹malate dehydrogenase的純化 71 第七節 綠竹malate dehydrogenase生化性質探討 74 7.1 最適反應條件 74 7.2 最適反應溫度 74 7.3 最適反應pH 75 7.4 MDH動力學分析 76 第八節 綠竹malate dehydrogenase抗體製備 78 8.1 小白鼠免疫 79 8.2 免疫檢驗 80 第九節 綠竹筍malate dehydrogenase之純化 82 9.1 粗抽取及硫酸銨分劃 82 9.2 親和層析－Blue Sepharose CL-6B 84 9.3 快速蛋白質液相層析 (Fast Protein Liquid Chromatography, FPLC) 85 第三章 實驗結果 90 第一節 Escherichia coli BL21 (DE3) 表現系統之建立 90 1.1 聚合酶鏈鎖反應 (PCR) 90 1.2 pET-32a(+) 重組載體之建構、轉形與檢定 91 1.3 綠竹基因組 (genomic library) 之篩選 92 第二節 綠竹MDH之大量表現並純化 93 2.1 E. coli BL21 (DE3) 轉型株表現型之檢定 93 2.2 綠竹MDH最適誘導時間點之探討 93 2.3 綠竹MDH之大量表現與純化 95 第三節 表現之綠竹MDH生化性質探討 96 3.1 最適反應溫度與活化能 96 3.2 最適反應pH 97 3.3 MDH之動力學分析 97 第四節 綠竹MDH抗體製備 98 第五節 綠竹筍MDH之純化 99 5.1 純化步驟之流程 99 5.2 粗抽取及硫酸銨分劃 100 5.3 Blue Sepharose CL-6B column 100 5.4 快速蛋白質液相層析 (Fast Protein Liquid Chromatography, FPLC) 101 第六節 綠竹筍MDH生化性質探討 104 6.1 最適反應溫度與活化能 104 6.2 最適反應pH 104 6.3 MDH之動力學分析 105 第七節 MDH生物資訊學分析 106 結果圖表集 107 第四章 討論與展望 138 附錄 142 參考文獻 153 問答集 159 | |
| dc.language.iso | zh-TW | |
| dc.title | 綠竹蘋果酸去氫酶之表現與檢定 | zh_TW |
| dc.title | Cloning, Expression and Characterization of Malate Dehydrogenase from Bamboo (Bambusa oldhamii) | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林棋財,楊健志 | |
| dc.subject.keyword | 綠竹蘋果酸去氫酶,大腸桿菌BL21 (DE3),gMDH,細胞分裂素氧化酶,/去氫酶,同質二元體, | zh_TW |
| dc.subject.keyword | BoMDH,E. coli BL21 (DE3),gMDH,BoCKX,homodimer, | en |
| dc.relation.page | 160 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-07-25 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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