Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3162
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dc.contributor林松池zh_TW
dc.contributor.author陳韻如zh_TW
dc.contributor.authorChen, Yun-Ruen_US
dc.contributor.other化學工程學系所zh_TW
dc.date2012en_US
dc.date.accessioned2014-06-06T05:31:18Z-
dc.date.available2014-06-06T05:31:18Z-
dc.identifierU0005-1308201217433600en_US
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Lee, 1996, Microcalorimetric studies of the interactions of imidazole with immobilized Cu(II): Effects of pH value and salt concentration, Journal of Colloid and Interface Science 183, 236-242. Wu, C. Y., S. Y. Suen, S. C. Chen, and J. H. Tzeng, 2003, Analysis of protein adsorption on regenerated cellulose-based immobilized copper ion affinity membranes, Journal of Chromatography A 996, 53-70. Zhang, S., and Y. Sun, 2002, Study on protein adsorption kinetics to a dye-ligand adsorbent by the pore diffusion model, J Chromatogr A 964, 35-46.en_US
dc.identifier.urihttp://hdl.handle.net/11455/3162-
dc.description.abstract本研究利用改質方式使尼龍菜瓜布表面改質(鹽酸水解) 、表面活化(戊二醛、己二胺、環氧氯丙烷) ,之後接上螯合劑和金屬離子 (Cu 2+ )後,即完成固定化金屬親和螯合吸附材之製備,並利用反應曲面法 (Response Surface Methodology, RSM) 探討鹽酸水解時間、戊二醛濃度、己二胺濃度對銅離子鍵結量之影響,以中心混成實驗找到吸附材合適的最佳參數條件。 從實驗結果發現尼龍菜瓜布經過改質後,當參數條件設為鹽酸水解時間 2.64 小時、戊二醛濃度 15.33%、己二胺濃度 0.19 M 時,銅離子鍵結量可達4.247 mmol/g 。在中心混成實驗部分,經由數學軟體統計回歸計算可得知,在陡升路徑實驗中選擇的範圍區域是正確的。 最後,利用製備出的吸附基材進行蛋白質 (2 – Epimerase) 吸附實驗,在蛋白吸附及脫附方面,判斷在 Flow through 部分的銅離子沉澱吸附了大部分的標的蛋白,導致實驗後續的 Wash、 Elution、 SDS-boiling 部分無法如預期清洗雜蛋白、脫附標的蛋白。zh_TW
dc.description.abstractIn this study, modified nylon sponges were prepared by first reacting original sponges sequentially with hydrochloric acid, glutaraldehyde, hexanediamine and epichlorohydrin. After practicing those procedures mentioned above, nylon sponges were then coupled with IDA (iminodiacetic acid) and loaded with Cu2+ to attain immobilized metal affinity (IMA) absorbents. The effect of hydrochloric acid hydrolysis time, glutaraldehyde concentration and hexanediamine concentration on the amount of chelated metal ion were analyzed by RSM (Response Surface Methodology) to decide the optimum values of independent factors for the best response value. Under optimnm conditions, hydrochloric acid hydrolysis time 2.64 hr, glutaraldehyde concentration 15.33%, and hexanediamine concentration 0.19 M, the amount of immobilized Cu2+ could reach up to 4.247 mmol/g. The results from RSM reveal the reliability for this study. At last, the results showed that there were Cu2+ precipitations in the buffer when adsorbing protein on prepared IMA absorbents. That is the reason for unpredicted experimental results.en_US
dc.description.tableofcontents中文摘要 i Abstract ii 目錄 iii 表目錄 vi 圖目錄 vii 第一章 諸論 1 第二章 文獻回顧 2 2-1 蛋白質純化 2 2-1-1 蛋白質簡介 2 2-1-2 蛋白質純化技術 3 2-2 固定化金屬親和層析法 5 2-2-1 簡介 5 2-2-2 固體相載體的組成 6 2-2-2-1 吸附基材 6 2-2-2-2 延伸臂 9 2-2-2-3 金屬螯合劑 10 2-2-2-4 過渡金屬離子 14 2-2-3固定化金屬層析和 polyhistidine tag 15 2-2-4影響吸附之因素 15 2-2-4-1 蛋白質結構 16 2-2-4-2 pH 值 17 2-2-4-3 鹽類及緩衝溶液 18 2-2-4-4 溫度 19 2-2-5 脫附的方法及影響因素 19 2-2-5-1 質子化 (protonation) 19 2-2-5-2 配位基交換法 (ligand exchange) 20 2-2-5-3 螯合消除法 (Chelate annihilation) 20 2-3 反應曲面法 22 2-3-1 反應曲面法之原理 23 2-3-2 反應曲面法實驗設計 24 2-3-2-1 二水準因子設計 (two-level factorial design) 24 2-3-2-2 陡升路徑法 (method of path steepest ascent, PSA) 26 2-3-2-3 中心混成設計 (central composite design, CCD) 28 2-3-2-4 反應曲面模式適切性之統計檢驗 29 2-4 研究系統簡介 32 2-4-1 尼龍 (Nylon) 32 2-4-2 戊二醛 (Glutaraldehyde, GA) 33 2-4-3 己二胺 (Hexanediamine, HEX) 33 2-4-4 環氧氯丙烷 (Epichlorohydrin, EPI) 33 2-4-5 反應曲面法 34 2-4-6 N - acetyl D - glucosamine 2 - epimerase (2 - Epimerase) 34 第三章 實驗藥品與儀器 37 3-1 實驗藥品 37 3-2 實驗儀器 39 第四章 實驗方法 40 4-1 吸附基材的製備 40 4-1-1 基材的清洗 40 4-1-2 基材表面的改質 40 4-1-2-1 尼龍水解 40 4-1-3 基材表面的活化 42 4-1-3-1 戊二醛 (Glutaraldehyde, GA) 42 4-1-3-2 己二胺 (Hexamethylenediamine, HEX) 42 4-1-3-3 環氧氯丙烷 (Epichlorohydrin, EPI) 42 4-1-4 基材表面的螯合 43 4-1-5 金屬離子的鍵結 43 4-1-6 清洗尼龍菜瓜布上弱鍵結銅離子 43 4-2 尼龍水解後表面形態觀察 (SEM) 47 4-3 反應曲面法設計之銅離子最大量 47 4-3-1 反應曲面法一階實驗設計 47 4-3-2反應曲面法陡升實驗設計 47 4-3-3反應曲面法二階實驗設計 52 4-3-4 銅離子鍵結量測定 52 4-4 酵素液的製備 54 4-4-1 菌種簡介 54 4-4-2 LB 固態培養基的製備 55 4-4-3 LB 液態培養基的製備 55 4-4-4 菌種培養方式 55 4-4-5 破細胞與可溶性蛋白質的收集 56 4-4-6 粗酵素液透析 57 4-5 銅離子最適化吸附脫附條件 57 4-6 蛋白質電泳分析 (SDS-PAGE) 58 4-6-1 樣品製作 58 4-6-2 膠體製備及蛋白質電泳 58 第五章 結果與討論 61 5-1 反應曲面法 61 5-1-1 二水準因子設計 (two-level factorial design) 61 5-1-2 陡升路徑法 (method of path steepest ascent, PSA) 62 5-1-3 中心混成設計 (central composite design, CCD) 62 5-1-4 檢測分析 69 5-2 表面形態觀察 (Scanning Electron Microscope, SEM ) 71 5-3 蛋白質吸附脫附 73 第六章 結論 76 第七章 參考文獻 78 附錄 85zh_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=U0005-1308201217433600en_US
dc.subject尼龍菜瓜布zh_TW
dc.subjectnylon spongeen_US
dc.subject反應曲面法zh_TW
dc.subject固定化金屬螯合親和吸附材zh_TW
dc.subjectresponse surface methodologyen_US
dc.subjectimmobilized metal ion affinity adsorbenten_US
dc.title以反應曲面法最適化改質尼龍菜瓜布作為固定化金屬螯合親和吸附材之研究zh_TW
dc.titleOptimization of modified nylon sponges as immobilized metal ion affinity adsorbents using response surface methodologyen_US
dc.typeThesis and Dissertationzh_TW
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