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研究生:曹立渟
研究生(外文):Tsao, Li-Ting
論文名稱:以分子內交聯提升紅藻紫菜色素 R-藻紅蛋白之穩定性
論文名稱(外文):Thermal stability enhancement of R-phycoerythrin from red alga Porphyra dentata via intramolecular crossslinking
指導教授:林泓廷
指導教授(外文):Lin, Hong-Ting
口試委員:許邦弘黃培安陳怡原賴信志林泓廷
口試委員(外文):Hsu, Pang-HungHwang, Pai-AnChen, Yi-YwanLai, Hsin-ChihLin, Hong-Ting
口試日期:2018-06-27
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:67
中文關鍵詞:藻膽蛋白藻紅蛋白分子內交聯穩定性蛋白質構型
外文關鍵詞:phycobiliproteinR-phycoerythrinintramolecular crosslinkingstability
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本論文 R-藻紅蛋白 (R-phycoerythrin, R-PE) 存在於紅藻中,為一種水溶性捕光色素螢光蛋白,蛋白質上接有藻膽素而帶有獨特顏色及螢光,在商業上具有著高度的經濟價值,可作為色素或作為螢光探針使用。已有許多文獻指出,藻紅蛋白容易環境因素如:高溫、pH 或光線影響使蛋白質變性因而其失去顏色及螢光等功能,因此本次實驗自長葉紫菜 (Porphyra dentata) 萃取純化 R-藻紅蛋白,探討是否可藉由交聯劑修飾藻紅蛋白之方法增強蛋白質結構以提升穩定性之目的。本次實驗首先將紫菜粉末以研缽處理五分鐘後進行超音波輔助萃取,得到濃度 0.11 mg/mL 及 純度 (purity index, PI) 0.88 之藻紅蛋白粗萃液,以陰離子交換層析法純化後可得到 PI 1.48 之 純化 R-PE,再使用膠體過濾層析法進行第二步純化後,PI 可提升至 5.15,使用電泳圖和 ESI-Q-TOF MS/MS 進行胜肽鑑定,顯示純化出的蛋白質為 R-PE 之 α 及 β 次單元。本次實驗使用交聯劑分別為戊二醛 (Glutaraldehyde, GA) 及二硫代琥珀酰亞胺基丙酸脂 (Dithiobis succinimidyl propionate, DSP ),其會與蛋白質中的一級胺產生穩定的共價鍵維持高穩定結構的 R-PE。光譜特性分析結果顯示,添加 1:5 之 GA,A565 與螢光強度分別提升 40% 及 75%;添加莫耳比率 1:50 的 DSP 可使螢光強度上升 1.6 倍,另外於可見光吸收圖譜結果中皆顯示修飾後 R-PE 之最大吸收光波長為 495, 535, 565 nm。熱穩定測試結果方面,於 50℃ 作用二十小時後,添加 1:5 GA 之 A565 下降幅度為 44%,控制組為 53%,螢光強度結果亦顯示添加 1:5 GA 之螢光強度高於控制組 50%;添加 1:50 及 1:5 DSP 於 50℃ 作用八小時之 A565 可維持 70%,螢光強度亦可維持 78% 及 70%,控制組已降至 50%以下。總體而言,交聯劑於藻紅蛋白中可提供額外共價鍵加強次單元間的結構穩定性,減緩因高溫造成次單元間的疏水交互力下降而導致多聚體的降解,進而提升藻紅蛋白的穩定性。
The purpose of this thesis phycobiliproteins (PBPs) are the major light-harvesting chromoproteins in some seaweeds, PBPs including phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). PE can be divided into four classes, based on their origin and absorption srectrum, includes R-PE (Rhodophyta), B-PE (Bangiales) and C-PE (Cyanophyta). Because R-PE could be used as a fluoredcent, it became more and more important in commercial sectors. But R-PE is susceptible to degradation by environmental factors, such as temperature, pH and light, leading to its degradation, thus limiting the application of R-PE. In this study, we purified R-PE from red alga Porphyra dentata, and increased R-PE stability via intramolecular crosslinking. R-PE was extracted by maceration in combination with ultrasonication. In biomass 5% extraction, the R-PE concentration and purity index (A565/280) achived 0.11 mg/mL, 0.88. Then, the extraction R-PE was purified by ion exchange chromatography and gel filtration. The purified R-PE showed absorption maxima at 565 nm, PI achieved was 1.48. Based on ESI-Q-TOF MS/MS result, the 20 kDa band on the SDS-PAGE was considered the α and β subunits. In this study, we used two crosslinker including glutaraldehyde (GA) and dithiobis succinimidyl propionate (DSP) to modify R-PE. GA and DSP could crosslink the primary amino group in the R-PE, in order to maintain the structure stability. The spectral analysis results showed the fluorescence intensity and A565 increased 40%, 75% after adding GA at a molar ratio 1: 5, and the fluorescence intensity increased 1.6 folds after adding DSP at a molar ratio 1:50. The U. V. absorption spectra results showed the maximum absorption was 495, 535, and 565 nm, and it was the same with native R-PE. On the thermal stability test, the R-PE, which was adding molar ratio 1:5 (R-PE/GA), incubated at 50°C for 20h. The result showed that A565 of R-PE treated with GA was decreased 44% but the control group was decreased 53%, and the fluorescence intensity increased 50% compared with the native R-PE. After treated at 50°C for 20h, the A565 could maintained 70% and the fluorescence intensity could also maintained 78% and 70% for the R-PE crosslink DSP with molar ratio 1:50 and 1:5. In conclusion, cross-linkers could provide additional covalent bonds in phycoerythrins to enhance the structural stability and decreasing the degradation of hydrophobic interactions between subunits due to high temperatures.
摘要 I
Abstract II
目錄 III
表目錄 VI
圖目錄 VII
附錄目錄 IX
縮寫表 X
壹、前言 1
貳、文獻整理 1
2.1 海藻 1
2.1.1 簡介 1
2.1.2 紫菜 2
2.1.3 海藻色素 2
2.2 藻膽蛋白 (phycobiliprotein) 3
2.2.1 組成 3
2.2.2 作用機制 4
2.2.3 經濟價值 4
2.2.4 影響藻膽蛋白降解的因子 6
2.2.4.1 溫度 6
2.2.4.2 酸鹼 6
2.2.4.3 光照 6
2.3 藻紅蛋白 (phycoerythrin) 7
2.3.1 簡介 7
2.3.2 組成 7
2.3.3 定量方式 7
2.3.4 抗氧化能力 8
2.5 提高蛋白質穩定之方法 8
2.5.1 蛋白質固定化: 8
2.5.2 添加穩定劑: 9
2.5.3 化學修飾 9
2.6 共價連結法 (Cross-linking) 9
2.6.1 原理 9
2.6.2 來源 10
參、實驗設計 11
肆、實驗材料與方法 12
4.1 實驗材料 12
4.1.1 藻體來源 12
4.1.2 化學藥品 12
4.1.3 試劑套組 13
4.1.4 實驗器材與耗材 13
4.2 實驗方法 14
4.2.1 原料前處理 14
4.2.2 萃取藻膽蛋白 14
4.2.3 蛋白質純化 15
4.2.4 蛋白質特性分析 16
4.2.5 藻紅蛋白與交聯劑的交聯反應 19
4.2.6 穩定性試驗 19
4.2.6.1 溫度 19
4.2.7分析方法 20
伍、結果與討論 21
5.1 最適萃取條件 21
5.2 藻紅蛋白的分離純化 22
5.2.1 離子交換樹脂純化 22
5.2.2 粒徑排阻層析法 23
5.2.3 藻紅蛋白之胜肽鑑定 23
5.3. 探討交聯劑對於R-藻紅蛋白之安定性影響 24
5.3.1 R-藻紅蛋白交聯戊二醛 24
5.3.2 R-藻紅蛋白交聯二硫代琥珀酰亞胺基丙酸脂 26
5.3.3討論 28
陸、結論 30
柒、參考文獻 31
捌、圖表 37
玖、附錄 63
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