天然水解酶具有高催化效率以及受質選擇性等優點，然而其易受環境影響因而解摺疊失去活性，由於天然水解酶主要由胺基酸所組成，因此近年來許多研究參考天然酵素活性中心胺基酸序列，並利用各式蛋白質結構作為骨架設計人工水解酶，然而此些人工水解酶催化能力仍不及天然酵素。由於膠原蛋白為人體中含量豐富的蛋白質，加上其特殊的三股螺旋結構，因此在本研究中，我們嘗試利用膠原蛋白模擬胜肽作為骨架，並加入天然酵素中常見的催化二聯體或三聯體，觀察此種胜肽作為人工水解酶的催化能力，我們使用CD光譜鑑定所設計的胜肽結構和熱穩定性，以及使用TEM觀察所形成的大型結構樣貌，最後利用UV-Vis光譜研究胜肽催化效率。在第一部分實驗中，我們嘗試置入不同的胺基酸組合，並改變所置入的胺基酸位置，結果發現經由置換的膠原蛋白模擬胜肽仍保有一定的穩定性，說明此胜肽結構適合作為人工解酶骨架，並且發現藉由三股螺旋結構能使催化二聯體更加靠近使催化效率提升；在第二部分實驗中，我們利用尾端Fmoc基團或是金屬離子使胜肽形成大型結構，並觀察胜肽所組成的大型結構其催化能力，結果發現由於Fmoc基團的存在能使置入胺基酸彼此靠近作用力提升因而增進催化能力，然而金屬離子的加入可能使其結構變得過於緊密因而遮蔽活性中心，使催化效率降低。我們利用膠原蛋白模擬胜肽做為骨架仿效天然水解酶，除了具有良好的穩定性，且對於較疏水的受質具有較好的催化能力，雖然整體催化效率仍不及天然水解酶，但整體胺基酸序列的設計能做為未來設計人工水解酶良好的參考。

Natural hydrolases have drawn much attention due to their high catalytic activity and chemical specificity. However, environmental change may make these enzymes unfold and lose their activities. Since the composition of natural hydrolases is amino acids, many studies have employed different types of peptide structures and used the amino acid sequences in the active sites of natural enzymes to construct artificial enzymes. Nevertheless, the catalytic efficiency of theses designed hydrolases cannot be as excellent as the natural enzymes. Collagen is the most abundant protein in human body and has a unique triple helica structure. In this study, we utilized collagen-mimetic peptides (CMPs) as our scaffolds to design artificial hydrolases. We used circular dichroism (CD), transmission electron microscopy (TEM), and UV-Vis spectroscopy to characterize the structure and measure the catalytic efficiency of a series of peptides with various arrangements of catalytic residues. We also studied the influence of the location of active site in CMPs on catalytic activity. In the first part, the designed CMPs could form stable triple helices, providing a stable skeleton for artificial hydrolases. Our results showed that the catalytic dyad in the triple helix could strongly interact with each other and exhibit a better catalytic efficiency. In the second part, we use Fmoc group and metal ions to promote the peptides to assembly into supramolecular structures. The results showed that the interaction within the catalytic dyad may be stronger with the assistant of Fmoc groups and improve the catalytic activity. However, the addition of metal ions may make structures become too compact to expose the active site, leading to the decrease of catalytic efficiency. Although the catalytic efficiency of our designed CMPs cannot be as good as that of natural enzymes, this study provides useful information for the development of artificial hydrolases in the future.

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