Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3487
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dc.contributor.advisor林松池zh_TW
dc.contributor.author何立凡zh_TW
dc.date2002zh_TW
dc.date.accessioned2014-06-06T05:32:02Z-
dc.date.available2014-06-06T05:32:02Z-
dc.identifier.urihttp://hdl.handle.net/11455/3487-
dc.description.abstract本研究利用自行合成的金屬親和吸附材將酵素(D-hydantoinase)純化及固定於吸附基材上,並直接應用於連續式反應操作希望建立一個可以同時純化及固定化酵素並可確實應用於反應操作的系統。 實驗材料選取矽膠為基材,因其表面具有OH基所以易於化學改質且機械強度夠,菌種取自於中興大學分生所許文輝教授成功發展的基因重組E.coil,利用基因重組技術,使E.coil所生產D-hydantoinase尾端均帶有六個組氨酸(histidine),如此即可以金屬親和吸附法純化之。 實驗結果發現雖然純化的效果不若商用純化膠體(Ni-NTA)來的好,且自行合成的吸附材上吸附較多的雜蛋白質,但在反應操作卻得到不錯的結果。不僅沒有其他的副反應產生且固定後的酵素最適反應溫度與pH值亦提昇。zh_TW
dc.description.abstractSeveral silica-based immobilized metal affinitychromatography (IMAC) adsorbents with different ligand densities(3-Glymocidoxyprop- -yltrimethoxysilane-Iminodiacetic acid to Iminodiacetic) were prepared for the purification of proteins and immobilization of enzymes. The IM- -AC adsorbents prepared with a ratio of 1:5 exhibit optimal binding cap- acity for His-tagged proteins and minimal nonspecific adsorption. Results of SDS-PAGE indicate that the adsorbent can be used for the purification of His-tagged D-hydatoinase(DHTase) for crude cell lysate in a single step process with a purify above 90%, comparable to commercial agarose -based IMAC adsorbents. Due to it's high mechanical strength, the silica- -based IMAC adsorbents are particularly well suited for industrial operations. A packed-bed bioreactor with the aforementioned IMAC adsorbent,loaded with DHTase was used forthe continuous bioconversion of D,L-p-HPH to N-carbamoyl-D-hydroxyphenylglicine. Under optimal conditions, 60℃,pH 8,a conversion of 60% was observed at a residue time of 20 mins. In this study ,we successful demonstrate that a packed- -bed bioreactor containing silica-based IMAC adsorbents can be employed for the purification and immobilization of His-tagged enzymes.en_US
dc.description.tableofcontents摘要………………………………………….………………..Ⅰ目錄………………………………………….………………..Ⅱ 圖目錄………………………………………….…………….Ⅲ 表目錄……………………………………………….……….Ⅳ 第一章 緒論…………… ……………………….………….1 第二章 文獻回顧…………………………….……..……..2 2-1金屬親和吸附法…………………………..……………2 2-2酵素固定化………………………………….………..5 2-3β-內醯胺(β-lactam)系列胺基酸…………………….7 2-4右旋乙內醯尿脢(D-hydantoinase)…………………..9 第三章 實驗藥品與儀器……………………….………..10 3-1實驗藥品………………………………….………..10 3-2實驗儀器………………………………….………..11 第四章 實驗方法與步驟………………………….……..12 4-1.酵素液之製備……………………………….……..12 4-2.矽膠膠體之合成與基本性質測試…………….…….15 4-3.酵素固定化…………………………………….…..17 4-4.批次反應…………………………………….……..19 4-5.連續式反應…………………………….…………..22 第五章 實驗結果與討論………………………………….26 5-1.金屬親和吸附材基本性質的量測……….………….26 5-2. 酵素固定化………………………………………..27 5-3. 批次反應…………………………………………..29 5-4. 連續式反應………………………………………..30 第六章 結論………………………………………………..33 第七章 參考文獻……….………………………………….34zh_TW
dc.language.isozh_TWzh_TW
dc.publisher化學工程學系zh_TW
dc.subjectIMACen_US
dc.subject金屬親和吸附zh_TW
dc.subjectHis-tagged proteinen_US
dc.subjectprotein purificationen_US
dc.subjectenzyme immobilizationen_US
dc.subject蛋白質純化zh_TW
dc.subject酵素固定化zh_TW
dc.title金屬親和吸附材於蛋白質純化及酵素固定化之應用zh_TW
dc.typeThesis and Dissertationzh_TW
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.fulltextno fulltext-
item.languageiso639-1zh_TW-
item.grantfulltextnone-
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