近年來，許多研究將簡單的胜肽序列設計成人工水解酶，期望能模擬天然水解酶的活性與選擇性。儘管常見的天然水解酶結構及序列已在文獻中被報導，組成活性中心的胺基酸序列與位置可以作為設計上參考，然而設計出同時具高催化活性及高受質結合性的水解酶仍是一大挑戰。在本研究中，我們設計以聚脯胺酸螺旋結構為主體的人工水解酶，並利用天然水解酶中催化二聯體、三聯體的概念，以及先前文獻中共同組裝的概念，研究聚脯胺酸結構對於催化效率的影響。我們以CD光譜鑑定所設計胜肽的結構及穩定性，並利用UV-Vis光譜以及一系列酯類受質研究人工水解酶的催化能力。在第一部分的實驗中，利用簡單的聚脯胺酸胜肽，我們發現PPII結構的完整性對於人工水解酶的催化效率有很重要的影響，並且去質子化過程也是影響人工水解酶的催化效率之重要因素；在第二部分的實驗中，利用膠原蛋白模擬胜肽，我們發現膠原蛋白三股螺旋的穩定性對於催化效率有很重要的影響，並且成功利用金屬與膠原蛋白模擬胜肽配位以提升催化效率。我們的研究說明在人工水解酶中，維持穩定三維結構及形成有效的催化中心才能使人工水解酶作為有效率的催化劑。本研究為首例將催化二聯體、三聯體引入聚脯胺酸結構的人工水解酶設計，並且催化效率優於部分文獻中具有大型奈米結構的水解酶設計，而我們所設計的人工水解酶也適合作為疏水性較高的大分子受質的催化劑。基於天然胺基酸所組成的人工水解酶具有較高的生物相容性與生物降解性，我們的設計也可在未來應用於生物系統中。

Recently, a number of simple oligopeptides have been developed as artificial enzymes for mimicking the activity and selectivity of natural hydrolases. Although the arrangement of amino acid residues in natural enzymes has been reported and their composition of active sites could provide a strategy for designing artificial enzymes, creating catalysts with both efficient binding and catalytic activity is still challenging. In this study, we designed a series of peptides with polyproline scaffold as artificial enzymes and also utilized the concept of catalytic dyad or triad and the co-assembly strategy. Their secondary structures were characterized by CD spectroscopy and their catalytic activities on ester hydrolysis were measured by UV-Vis spectroscopy using a series of p-nitrophenyl ester assay. In the first part, the results indicate that a well formed polyproline II (PPII) structure could result in a much higher catalytic efficiency. In addition, deprotonation steps in artificial enzymes are an important factor for obtaining a higher catalytic efficiency. In the second part, the results show that the triple helical stability of collagen-mimetic peptides could dramatically affect their catalytic efficiency. Furthermore, introducing zinc ions into collagen-mimetic peptides could mimic the active site in metalloenzymes and result in a higher catalytic efficiency. This is the first report of a functional dyad or triad engineered into a polyproline helix framework. Despite the fact that our designs are not as efficient as natural enzymes, the activity of our designs is still greater than some reported nanostructures. Our investigation has also revealed the necessity of maintaining a stable three-dimensional structure and a well-organized catalytic site for effective biocatalysts.

誌謝辭　I
中文摘要　II
Abstract　III
目錄　IV
圖目錄　VII
表目錄　X
第一章 緒論　1
1-1聚脯胺酸(Polyproline)　1
1-2膠原蛋白(Collagen)　3
1-3酯類水解反應(Ester hydrolysis reaction)　6
1-4天然水解酶的作用機制　8
1-5人工水解酶的設計　12
1-6研究動機　15
第二章 實驗部分　16
2-1實驗儀器　16
2-2實驗藥品　17
2-3圓二色光譜儀 (Circular dichroism spectrometer, CD)　20
2-4固相胜肽合成法 (Solid phase peptide synthesis, SPPS)　23
2-5化合物的合成、純化與鑑定　26
2-5-1 Fmoc-Tyr(tBu)-resin之合成　26
2-5-2 Fmoc-Pro-Hyp-Gly-OH之合成　27
2-5-3 p-Nitrophenyl-(2-phenyl)-propanoate (p-NPP)之合成　29
2-5-4 p-Nitrophenylmethoxyacetate (p-NPMA)之合成　30
2-6胜肽的合成、純化與鑑定　31
2-7光譜測量　33
2-7-1 UV光譜測量濃度　33
2-7-2 CD光譜測量　33
2-7-3催化水解活性實驗　34
2-8光譜數據分析　37
2-8-1 CD變溫實驗之數據處理　37
2-8-2活性實驗之數據處理　38
2-8-3利用1H NMR光譜量測胜肽pKa值實驗之數據處理　40
2-9計算模擬聚脯胺酸結構之研究方法　40
第三章 結果與討論　41
第一部分 設計聚脯胺酸短胜肽作為人工水解酶　41
3-1聚脯胺酸短胜肽之設計　41
3-2胜肽結構之鑑定　42
3-3對酯類水解反應的催化效率探討　45
3-4在不同pH值環境下的催化效率探討　49
3-5利用1H NMR光譜量測胜肽pKa值　51
3-6對酯類水解反應的選擇性探討　52
3-7計算模擬聚脯胺酸結構　56
第二部分 設計膠原蛋白模擬胜肽作為人工水解酶　59
3-8膠原蛋白模擬胜肽之設計　59
3-9膠原蛋白模擬胜肽三股螺旋結構之鑑定　60
3-10對酯類水解反應的催化效率探討　62
3-11與文獻中人工水解酶的催化效率比較　67
第四章 結論與展望　68
參考文獻　70
附錄　76

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