臺灣博碩士論文加值系統

高度孔隙性的苯乙烯與二乙烯苯交聯的共聚物(PS-DVB)顆粒常用來作為大小排擠層析與離子交換層析的固定相物質。若要應用於生化用途，例如蛋白質層析與酵素固定化，多孔隙結構的高分子顆粒則會有擴散限制，因此本研究致力於開發只有表層孔隙結構的共聚物顆粒，期望在生化用途上發揮更好的功效。
本研究利用多步驟腫脹聚合法來製備PS-DVB顆粒，探討在製備高分子造孔源(polymeric porogen)顆粒與進行共聚合時，苯乙烯用量與甲苯用量對最後所合成之顆粒在型態與孔洞生成的影響。並將Candida rugosa脂解酵素固定於所製備的載體上，探討在有機溶劑中的熱穩定性。
實驗結果發現在製備高分子造孔源顆粒時苯乙烯的用量與進行共聚合時甲苯的用量增多時，所合成出的顆粒具有較高的比表面積，且顆粒粒徑分佈尚稱均一。顆粒的特性如粒徑分佈及孔洞特性可經SEM觀測與比表面積分析儀量測得到。C. rugosa脂解酵素固定於所製備PS-DVB顆粒之後，最佳活性溫度提高，而且在有機溶劑中熱失活速率常數有明顯下降。但與固定於聚丙烯顆粒比較，發現第一階段失活之後，殘留活性較低，可能原因是固定於PS-DVB之後，酵素構形已有所改變。

Highly porous and cross-linked styrene-divinylbenzene copolymer (PS-DVB) particles have been widely used as the stationary phases for size exclusion and ion-exchange chromatography. For biological applications, for example, protein chromatography and enzyme immobilization, the porous structure of the particles could lead to diffusional limitation. In this study, the PS-DVB particles having a porous structure with large pores near the periphery of the particles were prepared for biological applications.
Styrene-divinylbenzene copolymer particles were prepared via a method of multi-step swelling and polymerization in this research. The influences of the amount of styrene used for preparing polymeric porogen particles and toluene used for copolymerization on the particle morphology and porous structure were studied. The prepared PS-DVB particles then were used for immobilization of Candida rugosa lipase. The thermostability of resultant immobilized lipase in organic solvent was also studied.
Increasing the amounts of the styrene and toluene, respectively, used for preparing polymeric porogen particles and copolymer could result in copolymer particles having a higher specific surface area and uniform size distribution, according to the results from Micromeritics (ASAP 2000) and the photographs of SEM. When C. rugosa lipase was immobilized on PS-DVB, the optimum temperature for enzymatic activity was increased and thermodeactivation in organic solvent was slowed down. However, in comparison with that immobilized on PP, the immobilized enzyme on PS-DVB retained a lower activity after the first stage deactivation due to a probable change in the conformation of enzyme molecule by immobilization.

目錄
中文摘要………………………………………………………………….I
英文摘要…………………………………………………………………II
目錄……………………………………………………………………..IV
圖目錄………………………………………………………………….VII
表目錄…………………………………………………………………..IX
第一章 緒論……………………………………………………………1
1.1研究緣由與目的………………………………………………..1
1.2文獻回顧………………………………………………………..2
1.2.1 PS-DVB顆粒的合成………………………………………..2
1.2.2 Polystyrene (PS)顆粒的製備………………………………..6
1.2.3酵素的固定化……………………………………………….7
1.3研究要旨與章節組織………………………………………….11
第二章 實驗理論……………………………………………………..13
2.1分散聚合法(Dispersion Polymerization)……………………...13
2.2懸浮聚合法(Suspension Polymerization)……………………..13
2.3孔洞形成的機構……………………………………………….15
2.4脂解酵素在有機溶劑中反應………………………………….19
2.4.1酵素在有機溶劑中的反應…………………………………19
2.4.2酵素失活模式………………………………………………19
第三章 實驗藥品、設備與方法………………………………………21
3.1實驗藥品……………………………………………………….21
3.1.1高分子顆粒的製備…………………………………………21
3.1.2酵素固定化…………………………………………………22
3.1.3酵素活性的檢驗……………………………………………22
3.1.4脂解酵素的水解反應及初速率的測定……………………22
3.1.5游離脂肪酸檢量線的製作及游離脂肪酸的測定…………23
3.2實驗設備……………………………………………………….23
3.3實驗步驟……………………………………………………….24
3.3.1 PS粒種的製備……………………………………………..24
3.3.2 Polymeric porogen顆粒的製備……………………………24
3.3.3苯乙烯與二乙烯苯共聚物顆粒的製備……………………25
3.3.4酵素的固定化………………………………………………25
3.3.5酵素活性的檢驗……………………………………………26
3.3.6酵素反應初速率的測定……………………………………27
3.3.7游離脂肪酸的測定…………………………………………28
3.3.8游離脂肪酸檢量線的製作…………………………………28
3.3.9水解反應之最適反應溫度…………………………………28
3.3.10酵素熱穩定性的研究……………………………………..30
第四章 結果與討論…………………………………………………..31
4.1 PS-DVB顆粒製備之結果…………………………………….31
4.1.1 PS粒種的合成……………………………………………..31
4.1.2 polymeric porogen顆粒的製備……………………………32
4.1.3腫脹polymeric porogen顆粒和styrene/DVB共聚合反應.34
4.1.4比表面積與顆粒直徑的關係………………………………49
4.2 Candida rugosa脂解酵素的固定化與其熱穩定性…………..52
4.2.1 Candida rugosa脂解酵素的固定化……………………….52
4.2.2自由態與固定態Candida rugosa脂解酵素的最適水解溫
度…………………………………………………………..55
4.2.3以序列型態模式描述Candida rugosa脂解酵素的熱失活
及失活活化能……………………………………………..58
第五章 結論與建議……………………………………………………65
參考文獻………………………………………………………………..68

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