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研究生: 吳保羅
BAU-LUO WU
論文名稱: 製備明膠/藍藻蛋白以戊二醛交聯之薄膜
Preparation of gelatin/cyanophycin film cross-linked with glutaraldehyde
指導教授: 曾文祺
Wen-Chi Tseng
口試委員: 方翠筠
none
陳秀美
none
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: 2012
畢業學年度: 100
語文別: 中文
論文頁數: 60
中文關鍵詞: 藍藻蛋白明膠
外文關鍵詞: cyanophycin, gelatin
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藍藻蛋白為一種非核醣體合成蛋白質存在於藍綠藻菌中,經由基因重組後利用大腸桿菌來生產藍藻蛋白,其結構為aspartic acid為骨架arginine與lysine為側鏈組成。明膠主要來源為動物的皮膚、骨骼與結締組織,由不同的胺基酸形成龐大且複雜的多肽分子再以不同方式排列形成三股螺旋。
藍藻蛋白與明膠皆具有良好的生物相容性,近幾十年明膠已廣泛利用於生醫材料上,而藍藻蛋白因機械性質較差還未被廣泛使用,本研究希望藉由戊二醛將明膠與藍藻蛋白薄膜進行交聯反應,提高藍藻蛋白的應用性。
首先將藍藻蛋白與明膠混合溶液於鐵氟龍片上風乾成膜後利用戊二醛進行交聯,將交聯過後的薄膜取下用萬能試驗機測試其機械性質,結果顯示隨著藍藻蛋白混合比例越高,其抗拉強度與伸長率有所下降。在膨脹率研究方面,將藍藻蛋白/明膠複合薄膜浸泡於37 ℃磷酸鹽緩衝溶液,測得其膨脹率隨著藍藻蛋白混合比例越高有下降趨勢,但仍保有一定膨脹率。最後以掃描式電子顯微鏡觀察薄膜表面,顯示出整體上薄膜表面完整且平坦,並沒有因為交聯或取膜過程中出現孔洞或明顯的裂痕。


Cyanophycin granule polypeptide (CGP) is a non-ribosomal protein found in cyanobacteria. CGP which is composed of aspartic acid (back bone), arginine and lysine (side chain) is produced with Escherichia coli through recombinant technology. Gelatin whose sources are animal skin ,bone and connective tissue is large and complex polypeptide of different amino acids, then arranged in different ways to form a triple helix.
Both of CGP and gelatin are well biocompatible. In recent decades, gelatin is widely used in biomaterials. However, CGP exhibits poor mechanical properties which limit its possible applications. In this study, to improve the applicability of CGP, we made CGP crosslink with gelatin by glutaraldehyde reaction.
In the beginning, we air-dried the mixed CGP/gelatin solution on teflon plate. After that we made the films crosslink with glutaraldehyde. The films were removed after crosslinking, and then we used universal test machine to test their mechanical properties. The result exhibits both tensile strength and elongation decreased with higher mixing ratio of CGP. In the swelling studies, the CGP/gelatin films were immersed in phosphate buffered saline at 37 ℃. We obtained the result that the swelling trend was downward with higher mixing ratio of CGP. Finally, using scanning electron microscopy to observe the surface morphology of the films, we found it smooth and homogeneous. They didn’t show cracks or holes when we removed the film froms teflon plate or during the crosslinking.

中文摘要 I Abstract II 目錄 III 圖目錄 VI 表目錄 VII 第一章 緒論 1 1.1 前言 1 1.2 研究動機 2 第二章 文獻回顧3 2.1 膠原蛋白 3 2.2 明膠介紹 8 2.3 明膠的結構與特性 9 2.3.1 穩定性 (stability) 9 2.3.2 溶解性 (solubility) 10 2.3.3 等電點 (isoelectric point) 10 2.3.4 黏度 (viscosity) 11 2.3.5 結構及組成 11 2.3.6 明膠強度 14 2.4 明膠生產製程 14 2.5 明膠的應用 16 2.5.1 生醫材料 16 2.5.2 藥物載體 17 2.5.3 食品 17 2.6 藍藻蛋白 18 2.6.1 藍藻蛋白簡介 18 2.6.2 藍藻蛋白應用與發展 21 2.7 戊二醛與lysine交聯反應機制 23 第三章 實驗 25 3.1 實驗材料 25 3.1.1 菌株 25 3.1.2 抗生素 25 3.1.3 化學藥品 25 3.1.4 實驗器具 26 3.2 實驗設備 27 3.3 藥品製備 28 3.3.1 Luria-Bertani Medium (LB medium) 28 3.3.2 Luria-Bertani Medium plate 28 3.3.3 Terrific Broth (TB) 29 3.3.4 Ampicillin 儲存溶液 29 3.3.5 Chloramphenicol儲存溶液 29 3.3.6 Isopropyl β-D-Thiogalactopyranoside儲存溶液 30 3.3.7 磷酸鹽緩衝溶液 (phosphate buffer solution) 30 3.4 實驗步驟 31 3.4.1 微生物培養 31 3.4.1.1 培養大腸桿菌於LB Medium plate 31 3.4.1.2 培養大腸桿菌於2 mL LB Medium 31 3.4.1.3 培養大腸桿菌於60 mL LB Medium 32 3.4.1.4 培養大腸桿菌於3 L發酵槽32 3.4.2 藍藻蛋白純化 33 3.4.2.1 水溶性藍藻蛋白純化 34 3.4.2.2 非水溶性藍藻蛋白純化 35 3.4.3 製備藍藻蛋白/明膠複合薄膜 36 3.5薄膜樣品物性測試 37 3.5.1薄膜厚度量測 (thickness measurements) 37 3.5.2 膨脹率量測 (swelling measurements) 37 3.5.3 以萬能試驗機 (universal test machine) 測試不同比例薄膜的力學性質 38 3.5.4 以掃描式電子顯微鏡 (scanning electron microscopy) 觀察薄膜表面形態 39 第四章 結果與討論 40 4.1 藍藻蛋白與明膠交聯成膜 40 4.2 薄膜顏色觀察 40 4.3 薄膜厚度量測 41 4.4 薄膜膨脹率量測 42 4.5 薄膜力學性質 43 4.6 薄膜表面形態 44 第五章 結論 45 參考文獻 46

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