臺灣博碩士論文加值系統

五胺基酮戊酸（5-ALA）是一種參與人體血紅素代謝的非蛋白氨基酸，目前已廣泛應用於農業和醫學領域。 5-ALA可以在驅動下轉化為光敏劑- PpIX，並釋放活性氧 (Reactive oxygen species, ROS)，是為光動力療法 (Photodynamic therapy, PDT)消除病原體或癌細胞的前驅藥。生物合成5-ALA是有效且環保的方法，但培養基中成份複雜較難直接使用，因此後續純化是必要的。在這項研究中，比較鹽酸，醋酸鈉及氨水對5-ALA的脫附效果，最終在pH 9.5，1 M的氨水中得到92%的回收率。隨後，加入活性碳進行脫色去除脫附液中的色素。接著以乙醇和丙酮二步法析出沉澱獲得純度85%的5-ALA結晶。隨後，使用0.25%的純化5-ALA對豪氏變形桿菌 (Proteus hauseri, P. hauseri), 嗜水氣單胞菌 (Aeromonas hydrophila, A. hydrophila), 仙人掌桿菌 (Bacillus cereus, B. cereus) 和金黃色葡萄球菌 (Staphylococcus aureus, S. aureus)進行光動力殺菌 (aPDT)，獲得100%殺菌效率證實了純化5-ALA的抗菌效果。同時，純化後的5-ALA也應用於促進藻類的生長並消除污染藻類培養液的嗜水氣單胞菌，在0.2%的5-ALA幫助下，去除了67.9%的嗜水氣單胞菌並提升了藻類的生長。
這項研究探討各種不同溶液對5-ALA的脫附效果，利用有機溶劑對5-ALA進行結晶；純化後5-ALA對三種病原菌達成100%殺菌效率，且5-ALA可促進藻類在細菌存在的培養環境不受細菌的影響，除了具有殺菌效率更提高產量，在5-ALA應用上開展了新的方向。

5-aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid involved in human heme metabolism, which has been broadly applied in the agriculture and medical fields nowadays. 5-ALA can be used for the elimination of pathogens or cancer cells by photodynamic therapy (PDT) owing to its conversion to photosensitizer and release reactive oxygen species (ROS). The biofabrication of 5-ALA is regarded as the most efficient and eco-friendly approach, but the complicated ingredient of medium affects 5-ALA purification, resulting in low recovery and high cost. In this study, HCl, sodium acetate and ammonia were examined to maximize the recovery of 5-ALA from cation exchange chromatography, resulting a 93% recovery in 1 M ammonia at pH 9.5. Afterward, the activated carbon was added for decolorization to further remove other pigments. Different organic solvents were compared for 5-ALA precipitation by 2-steps poor solvent method after being concentrated to 400 g/L by a rotary evaporator. The purify 5-ALA was verified to eliminate Proteus hauseri, Aeromonas hydrophilia (A. hydrophilia), Bacillus cereus and Staphylococcus aureus via anti-microbial PDT with 0.25% 5-ALA to reaching 100% killing rate, respectively. Surprisingly, the growth of microalgae Chlorella sorokiniana was significantly improved with 0.05% 5-ALA to against A. hydrophilia which is a common aquatic pathogen during the cultivation.

摘要 I
Extend Abstract II
誌謝 XI
目錄 XII
表目錄 XV
圖目錄 XVI
縮寫對照表 XVII
第一章 緒論 1
1.1 前言 1
1.2 研究與目的與架構 3
第二章 文獻回顧 5
2.1 五胺基酮戊酸 (5-ALA) 5
2.1.1 5-ALA的介紹 5
2.1.2 5-ALA的化學合成方法 5
2.1.3 5-ALA之生物合成方法 7
2.2 5-ALA純化 8
2.2.1 活性碳脫色 8
2.2.2 離子交換層析 10
2.2.3 析出沉澱 12
2.3 5-ALA之應用 14
2.3.1 在農業上的使用 14
2.3.2 在微藻上的應用 15
2.3.3 光動力治療 16
第三章 實驗材料與方法 19
3.1 實驗藥品 19
3.2 實驗儀器 21
3.3 菌株 22
3.4 溶液配製 23
3.5 實驗方法 25
3.5.1 菌株培養及保存 25
3.5.2 五胺基酮戊酸 (5-ALA)濃度測定 26
3.5.3 利用高效液相色譜（HPLC）進行胺基酸定量分析 28
3.5.3.1 胺基酸定量分析 28
3.5.4 利用離子交換層析和析出沉澱法純化5-ALA 29
3.5.4.1 離子交換樹脂前處理 29
3.5.4.2 吸脫附5-ALA 29
3.5.4.3 活性碳脫色 30
3.5.4.4 旋轉濃縮 30
3.5.4.5 有機溶劑析出結晶 30
3.5.5 5-ALA光動力殺菌 (5-ALA-aPDT) 30
3.5.5.1 光動力殺菌 30
3.5.6 5-ALA在微藻-菌共培養基殺菌實驗 31
3.5.6.1 Chlorella sorokinana (Cs)培養 31
3.5.6.2 5-ALA在微藻-菌共培養基的殺菌實驗 31
第四章 結果與討論 33
4.1 5-ALA離子交換層析的條件優化 33
4.1.1 不同策略放大培養5-ALA發酵液 33
4.1.2 不同溶液對5-ALA的脫附效率 35
4.1.3 以不同液體濃度及pH值優化5-ALA脫附條件 36
4.1.4 樹脂的重複使用測試 38
4.2 5-ALA析出結晶 38
4.2.1 5-ALA脫附液脫色 38
4.2.2 旋轉蒸餾提升5-ALA脫附液濃度 39
4.2.3 不同有機溶劑析出5-ALA結晶 40
4.2.4 二次結晶析出5-ALA 42
4.3 純化5-ALA結晶在光動力殺菌 (aPDT)的應用 43
4.3.1 田口L9直交法優化5-ALA aPDT操作條件 43
4.3.2 以純化5-ALA結晶進行Proteus hauseri殺菌 45
4.3.3 純化後5-ALA結晶對其他病原菌的抗菌效率 47
4.4 5-ALA在微藻中的應用 49
4.4.1 5-ALA於微藻-細菌共培養中的殺菌效率 50
第五章 結論與未來展望 53
5.1 結論 53
5.2 未來展望 55
第六章 參考文獻 56
附錄 66

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