Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3095
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dc.contributor林松池zh_TW
dc.contributor.author陳竑毅zh_TW
dc.contributor.authorChen, Hong-Yien_US
dc.contributor.other化學工程學系所zh_TW
dc.date2012en_US
dc.date.accessioned2014-06-06T05:31:06Z-
dc.date.available2014-06-06T05:31:06Z-
dc.identifierU0005-1508201211095800en_US
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dc.identifier.urihttp://hdl.handle.net/11455/3095-
dc.description.abstract利用大腸桿菌生產N-胺甲醯基-L型-胺基酸胺基水解酵素時,會因大量表現而產生內函體。利用Tris buffer直接溶解內函體後即可得到活性蛋白。其中以pH 9.0所回收得來的蛋白比活性0.40 U/mg為最高,但還是不如native蛋白(pH 8.0下比活性為1.58 U/mg,pH 9.0下為1.31 U/mg)。將native蛋白加入DTT後可發現比活性迅速降低,顯示雙硫鍵對於比活性具有重大影響。而從內函體中回收的可溶蛋白不具有雙硫鍵結構,所以缺乏正確的雙硫鍵結構可能是導致活性不如native蛋白的原因。 由於雙硫鍵對於蛋白活性具有重大影響,故在後續進行復性實驗時加入GSH/GSSH以幫助雙硫鍵的形成。在復性緩衝溶液中添加總濃度[GSH]+2[GSSG]=10 mM,比例[GSH]/2[GSSG]=4的情況下,可將可溶蛋白回收率從80.0%提升10.3%至90.3%。添加總濃度[GSH]+2[GSSG]=10 mM,比例[GSH]/2[GSSG]=10或8的情況下,則可將總活性從0.021 U提升76%至0.037 U。 整體而言,添加GSH/GSSH可確實幫助N-胺甲醯基-L型-胺基酸胺基水解酵素提升復性產率。zh_TW
dc.description.abstractRecombinant N-carbamoyl-L-amino acid amidohydrolase was over-expressed in Escherichia coli in the form of inclusion bodies. Active protein could be recovered from inclusion bodies by direct solubilization with Tris buffer. The largest specific activity of solubilized protein is 0.40 U/mg at pH 9.0, but it was still lower than native protein, 1.58 U/mg at pH 8.0 and 1.31 U/mg at pH 9.0. The result of adding DTT in native protein indicated that disulfide bond structures had significant impact on specific activity. The structures of solubilized proteins from inclusion bodies didn’t have disulfide bond structures, so lack of right disulfide bond structures may be the reason of lower specific activity. Because of the significance of disulfide bond structures, GSH/GSSH was added into the refolding system to assist formation of disulfide bonds. Recovery of soluble protein could be increased by 10.3% from 80.0% to 90.3% when the refolding buffer contained 10 mM of a 4:1 ratio of [GSH]/2[GSSG]. Total activity could be increased by 76% from 0.021 U to 0.037 U when the refolding buffer contained 10 mM of a 10:1 or 8:1 ratio of [GSH]/2[GSSG]. Overall, adding GSH/GSSG could enhance refolding result of N-carbamoyl-L-amino acid amidohydrolase.en_US
dc.description.tableofcontents中文摘要 i Abstract ii 目錄 iii 表目錄 v 圖目錄 vi 第一章 緒論 1 1-1 前言 1 1-2 研究動機 1 第二章 文獻回顧 3 2-1 蛋白質結構與摺疊 3 2-1-1 蛋白質結構 3 2-1-2 蛋白質摺疊 4 2-2 內函體 4 2-2-1 內函體的形成 4 2-2-2 內函體的回收與純化 6 2-2-3 內函體的品質 6 2-2-4 內函體溶解 6 2-3 蛋白質復性 8 2-3-1 復性方法 8 2-3-2 提高復性產率之方法 11 2-3-3 雙硫鍵的形成 14 2-4 L-N-carbamoylase介紹 15 第三章 實驗藥品與儀器 16 3-1 實驗藥品 16 3-2 實驗儀器 17 第四章 實驗方法 18 4-1 製備N-Carbamoyl-L-HPA 18 4-2 菌種的培養 19 4-2-1 LB固態洋菜培養基的製備 19 4-2-2 LB液態培養基的製備 19 4-2-3 菌種培養 19 4-3 菌體、粗酵素液與內函體的回收 20 4-3-1 菌體的回收 20 4-3-2 粗酵素液與內函體的回收 20 4-4 native蛋白的分析 20 4-4-1 粗酵素液的純化 20 4-4-2 pH值對native蛋白活性的影響 21 4-4-3 DTT對native蛋白活性的影響 21 4-5 內函體的分析 22 4-5-1 pH值與DTT對內函體溶解度的影響 22 4-5-2 內函體中可溶蛋白的純化與分析 22 4-5-3 內函體的變性 22 4-5-4 初始濃度與稀釋倍率對蛋白質回收率的影響 23 4-5-5 GSH/GSSG對內函體復性的影響 23 4-6 分析方法 23 4-6-1 蛋白質濃度分析 23 4-6-2 電泳分析 24 4-6-3 蛋白質圓二色光譜測試 26 4-6-4 L-N-carbamoylase活性分析 26 第五章 結果與討論 28 5-1 N-Carbamoyl-L-HPA製備之探討 28 5-2 L-N-carbamoylase的表現 28 5-3 從內函體回收可溶蛋白 28 5-3-1 pH值與DTT對內函體溶解度的影響 28 5-3-2 可溶蛋白的活性探討 29 5-3-3 可溶蛋白的純度分析 30 5-3-4 可溶蛋白的二級結構分析 30 5-3-5 雙硫鍵對活性的影響 31 5-4 內函體的復性 32 5-4-1 變性劑對內函體溶解度的影響 32 5-4-2 稀釋倍率對蛋白質回收率的影響 33 5-4-3 GSH/GSSH對蛋白質回收率的影響 33 第六章 結論 52 第七章 參考文獻 54 附錄 計算式定義 58zh_TW
dc.language.isozh_TWen_US
dc.publisher化學工程學系所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1508201211095800en_US
dc.subjectN-胺甲醯基-L型-胺基酸胺基水解酵素zh_TW
dc.subjectrefoldingen_US
dc.subject穀胱甘肽zh_TW
dc.subjectN-carbamoyl-L-amino acid amidohydrolaseen_US
dc.subjectGSHen_US
dc.subjectGSSGen_US
dc.title從內函體回收N-胺甲醯基-L型-胺基酸胺基水解酵素zh_TW
dc.titleRecovery of N-carbamoyl-L-amino acid amidohydrolase from inclusion bodiesen_US
dc.typeThesis and Dissertationzh_TW
item.grantfulltextnone-
item.openairetypeThesis and Dissertation-
item.languageiso639-1zh_TW-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.cerifentitytypePublications-
item.fulltextno fulltext-
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