溫度對經聚乙二醇雙性高分子修飾蛋白酶之效應｜國立臺灣科技大學博碩士論文系統

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研究生：	林亞昀 Ya-Yun Lin
論文名稱：	溫度對經聚乙二醇雙性高分子修飾蛋白酶之效應 The Effects of Temperature on α-chymotrypsin modified by the Poly(ethylene glycol)-Containing Amphiphilic Graft Copolymers
指導教授：	陳崇賢 Chorng-Shyan Chern
口試委員:	李振綱 Cheng-Kang Lee 邱信程 Hsin-Cheng Chiu
學位類別：	碩士 Master
系所名稱：	工程學院 - 化學工程系 Department of Chemical Engineering
論文出版年：	2010
畢業學年度：	98
語文別：	中文
論文頁數：	91
中文關鍵詞：	胰凝乳蛋白酶、酪蛋白
外文關鍵詞：	α- Chymotrypsin, Azocasein
相關次數：	點閱：202 下載：3
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　　本研究是利用酯交換反應合成一雙性高分子，以具有酸鹼敏感性的聚丙烯酸(PAAc)作為主鏈，聚乙二醇(PEG)作為高分子之側鏈，此雙性高分子則作為胰凝乳蛋白酶（α-chymotrypsin）接枝之載體。
實驗針對酵素修飾前後、接枝高分子含量多寡以及在不同的溫度、pH值環境下，分別以N-Benzoyl-L-Tyrosine ethyl ether（BTEE）小分子基質和Azocasein大分子基質，與酵素進行催化水解反應比較。
實驗結果發現，於小分子基質反應下，高分子載體中PEG含量增加時，酵素的相對剩餘活性會隨之增加，但於大分子基質反應下，因為位阻效應，隨著載體中的PEG含量增加，酵素的相對剩餘活性會隨之降低；而隨著熱處理時間增長，酵素相對剩餘活性皆會降低。經雙性高分子修飾後的酵素，因為高分子中酸（~COOH )的增加，使之能在高pH值中與鹼中和，使相對剩餘活性趨於穩定；於低pH值環境下，因為高分子捲曲、官能基失活，酵素的相對剩餘活性表現皆為不佳。藉由大、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。

Amphiphilic copolymers comprising poly(acrylic acid)(PAAc) as the backbone and monomethyl poly(ethylene glycol)(mPEG) as the grafts were synthesized and characterized. The copolymers were used for conjugation with different amounts of α-chymotrypsin. The activities of α-chymotrypsin before and after incorporating into the graft copolymers toward high and low molecular weight substrates were studied.
N- Benzoyl-L-Tyrosine ethyl ether (BTEE) was used as a low molecular weight substrate and Azocasein was used as a high molecular substrate. Residual activity increased with increasing the pH and the mPEG content when BTEE was used, but it decreased with increasing the temperature and the mPEG amount when Azocasein was used due to the steric effect associated with the excessive amounts of mPEG. In addition, enzyme’s residual activity was reduced with temperature increment.

第一章	緒論.................................................1
1-1 研究背景與目的............................................1
2-2 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高分子微胞材料之選擇.......................................5
2-3聚丙烯酸之特性............................................ 7
2-4聚乙二醇之特性............................................ 8
2-5 α-chymotrypsin之性質.....................................9
2-6 Azocasein之性質..........................................13
2-7酵素固定化................................................14
2-7-1固定化酵素之性質........................................14
2-8蛋白質分解酵素對蛋白質之催化作用..........................15
  2-8-1酵素催化機制..........................................17
第三章	實驗藥品與方法......................................19
  3-1實驗藥品................................................19
  3-2實驗儀器及設備..........................................24
  3-3實驗流程................................................26
  3-4合成步驟................................................27
   3-4-0 雙性高分子合成與蛋白質接枝反應......................27
   3-4-1 N-acryloxysuccinimide 之合成........................29
          3-4-1-1 NAS鑑定....................................30
   3-4-2 Poly N-acryloxysuccinimide 之合成...................31
   3-4-3 mPEG-NH2 之合成.....................................32
          3-4-3-1 mPEG-Cl 之改質.............................32
          3-4-3-2 mPEG-N3 之合成.............................34
          3-4-3-3 mPEG-NH2 之合成............................34
          3-4-3-4 mPEG-NH2 轉化率測定........................35
   3-4-4 PNAS-mPEG 之接枝反應................................36
   3-4-5 PNAS-mPEG 之水解反應................................37
   3-4-6 PNAS-mPEG與α-chymotrypsin 之接枝反應...............39
  3-5雙性高分子之性質鑑定....................................41
   3-5-1 Poly(NAS) 之性質分析................................41
   3-5-2接枝高分子之組成測定.................................41
   3-5-3接枝高分子之官能基測定...............................42
   3-5-4 PAAc-mPEG-ChT之性質鑑定.............................42
          3-5-4-1 Bio-Rad蛋白質濃度分析......................42
          3-5-4-2 蛋白質定性分析.............................43
   3-5-5活性分析.............................................44
          3-5-5-1 小分子活性分析.............................44
          3-5-5-2大分子活性分析..............................45
第四章	結果與討論..........................................46
  4-1 1H-NMR 光譜分析........................................46
   4-1-1反應單體之 1H-NMR 光譜分析...........................46
   4-1-2雙性高分子之組成測定.................................48
  4-2 FTIR光譜分析...........................................52
  4-3 Bio-Rad蛋白質濃度分析..................................59
   4-3-1酵素固定化高分子後各組成之分析.......................60
  4-4 SDS-PAGE分析...........................................61
  4-5活性分析................................................62
第五章	結論................................................73
第六章	參考資料............................................76
附錄A 高分子性質之計算.......................................79
附錄B .......................................................86

                                

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