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研究生:陳怡雯
研究生(外文):Yi-Wen chen
論文名稱:含聚乙二醇雙性高分子之製備與其固定化酵素之研究
論文名稱(外文):Preparation of Poly(ethylene glycol)-Cotaining Amphiphilic Graft Copolymers and Their Effect on the Immobilized enzyme
指導教授:陳崇賢陳崇賢引用關係
指導教授(外文):Chorng-Shyan Chern
學位類別:碩士
校院名稱:國立臺灣科技大學
系所名稱:化學工程系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:96
中文關鍵詞:酵素固定化雙性高分子胰凝乳蛋白酶牛血清蛋白
外文關鍵詞:enzyme immobilizationamphiphilic copolymerα-chymotrypsinbovine serum albumin
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  • 被引用被引用:2
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為了增加酵素的穩定性、方便酵素的回收利用,常會運用酵素固定化的技術改善之。本研究將探討酵素固定化於高分子載體後,造成酵素分子與基質相互作用之催化能力的改變。本實驗以酯交換反應合成一雙性高分子,是以酸鹼敏感性聚丙烯酸 (PAAc) 為主鏈,聚乙二醇(PEG) 作為高分子之側鏈,此雙性高分子則為酵素 (α-chymotrypsin) 固定化之載體。
本論文針對胰凝乳蛋白酵素於固定化高分子前後,以及固定化高分子含量多寡行催化反應之比較,分別以N-Benzoyl-L-Tyrosine ethyl ether (BTEE) 小分子基質及BSA大分子基質,與酵素進行催化水解反應。結果發現當高分子載體中PEG含量增加,則酵素的剩餘活性相對提高,尤其於pH 9下有較高的活性存在;而在與BSA基質作用下,酵素會因固定化高分子中PEG的含量,影響催化作用的分解速率,當在pH 7環境時,於特定PEG含量下其BSA基質被水解速率明顯增加,然而一但PEG含量持續增加反而因立體排斥效應,使酵素不易與大分子基質結合因而影響水解速率。本研究乃經由大、小基質於催化水解反應下之現象差異,了解酵素、載體與基質間各種作用力的消長。
Enzyme immobilization is one of the most important techniques for increasing the stability of enzyme and recycling.
In this study, it is intended to investigate the activity of enzyme after immobilization toward high and low molecular weight substrates. Amphiphilic copolymer comprising poly(acrylic acid)(PAAc) as the backbone and monomethoxy poly(ethylene glycol)(mPEG) as the grafts were synthesized and characterized. The copolymers were used for conjugation with different amounts of α- chymotrypsin as a pH-sensitive immobilized enzyme system. N-Benzoyl-L-Tyrosin ethyl ether was used as a low molecular weight substrate whereas bovine serum albumin was used as a high molecular substrate. Residual activity increased with increasing the mPEG amount and pH. The cleavage rate of bovin serum albumin was accelerated obviously for the experiments carried out in the pH 7 buffer solution. However, the excess amounts of mPEG would induce steric effect, thereby leading to slow cleavage rate.
目 錄
第一章 緒論..................................................1
1-1 研究背景與目的...........................................1
1-2 研究內容簡介.............................................2
第二章 文獻回顧..............................................3
2-1 雙性高分子...............................................3
2-2 高分子微胞在藥物控制釋放上之應用.........................4
2-3 高分子微胞材料之選擇.....................................7
2-4 聚丙烯酸之特性...........................................8
2-5 聚乙二醇之特性...........................................9
2-6 α-chymotrypsin之性質....................................10
2-7 酵素固定化..............................................13
2-8 蛋白質分解酵素對蛋白質之催化作用........................13
第三章 實驗部分.............................................17
3-1 實驗原料................................................17
3-2 實驗儀器及設備..........................................22
3-3 合成步驟................................................24
3-3-0 雙性高分子合成與蛋白質接枝反應.......................24
3-3-1 N-acryloxysuccinimide之合成..........................26
3-3-2 Poly N-acryloxysuccinimide之合成.....................28
3-3-3 mPEG-NH2之製備.......................................29
3-3-4 PNAS-mPEG之接枝反應..................................32
3-3-5 PNAS-mPEG與α-chymotrypsin之接枝反應..................33
3-3-6 PNAS-mPEG之水解反應..................................35

3-4 雙性高分子之鑑定........................................37
3-4-1 Poly(NAS)之鑑定…....................................37
3-4-2 接枝高分子之組成測定.................................37
3-4-3 接枝高分子之官能基測定...............................38
3-4-4 PAAc-mPEG-ChT之性質鑑定..............................38
3-4-5 色胺酸螢光光譜分析....................................39
3-4-6 活性分析..............................................39
3-4-7 PAAc-mPEG-ChT 對蛋白質基質之動力學測…40
第四章 結果與討論...........................................42
4-1 FTIR光譜分析............................................44
4-2 1H-NMR光譜分析..........................................48
4-3 Bio-Rad 蛋白質濃度分析..................................52
4-4 凝膠滲透層析(GPC).....................................54
4-5 SDS-PAGE分析............................................56
4-6 色胺酸(Trp)螢光光譜分析.................................57
4-7 活性分析…………………………............................60
4-8 酵素固定化產物與與牛血清蛋白之催化反應…................64

第五章 結論與建議...........................................71
5-1 結論....................................................71
5-2 建議....................................................72
第六章 參考文獻.............................................73
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