以酵素法萃煉微藻污泥之長鏈均質聚磷酸鹽

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林依萱(Yi-Hsuan Lin) 查詢紙本館藏

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論文名稱

以酵素法萃煉微藻污泥之長鏈均質聚磷酸鹽
(Cell-free enzymatic synthesis of insoluble and homogeneous long‐chain polyphosphate from microalgal biomass)

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★ 利用一鍋式高溫蛋白酶串聯反應將豆渣升級再造為生物永續製造之蛋白質原料

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至系統瀏覽論文 (2025-6-30以後開放)

摘要(中)

磷是生命中必要的元素，然而磷礦（Phosphate rock, PR）卻是一種有限且不可再生的重要礦產資源。隨著科技日益進步與人口逐年增長，磷資源的開採與應用也隨之增加，過去數據統計約80%的磷礦資源被運用於肥料與清潔劑上。然而，過量的磷排放至環境水體中容易導致水體優養化。因此，本研究首先利用微藻從豬場廢水中回收磷，然後使用磷酸轉移酶級聯生成不溶性和均質的長鏈聚磷酸鹽（polyphosphate, polyP）。polyP功能因鏈長而異，短鏈polyP（10–100-mer）可促進傷口癒合並促進骨再生；中鏈polyP（100–300-mer）具有抗菌特性（例如：M. tuberculosis）及抗病毒作用（例如：Covid）；另一方面，長鏈polyP（300–1,300-mer）可用於製造人工骨縫線，促進骨礦化，polyP具有生物醫學材料之高經濟價值。我們成功地從微藻中萃取了不同長度的polyP，然後將萃取的polyP分別通過聚磷酸激酶和肌酸激酶轉化為不溶性和均質的長鏈polyP。由此產生的不溶性polyP顆粒可以藉由過濾純化。最後，透過95℃非生物水解方式縮短長鏈polyP，以達到控制生成特定長度polyP之目的。本研究成功以酵素法萃煉微藻為高價值且均質的polyP，這是一種新型循環P生物經濟模型，該模型成功地將生物質廢物轉化為高價值的生物醫學材料。本研究不僅符合聯合國提出永續發展目標中的第6項「淨水及衛生」、第9項「工業化、創新及基礎建設」第12項「責任消費及生產」，也能解決磷礦資源不足及水體優養化等環境問題。

摘要(英)

Phosphorus is an essential component of cells. The availability of phosphate rock, the primary source of phosphorus, is limited and non-renewable. In the past few decades, most phosphorus resources were used to generate fertilizers and cleaning agents. However, improper discharge of phosphorus led to the accumulation of phosphorus and eutrophication in aquatic environments. Therefore, this study first harnessed microalgae to recover phosphorus from piggery wastewater, and then generated insoluble and homogeneous long-chain polyphosphate (polyP) using a streamlined phosphotransferase cascade. The polyP function varies depending on the chain length. Short‐chain polyP (10–100-mer) enhances the healing of wounds and promotes bone regeneration. Mid‐chain polyP (100–300-mer) possesses antimicrobial properties (e.g., M. tuberculosis) and exhibits antiviral effects (e.g., Covid). On the other hand, long‐chain polyP (300–1,300-mer) is useful for creating artificial bone stitches, promoting bone mineralization, and has greater economic and practical value compared to crystals. We successfully extracted polyP mixtures with varying lengths from microalgae, and the extracted polyP mixture was then converted into insoluble and homogenous long-chain polyP by polyphosphate kinase and creatine kinase, respectively. The resulting insoluble polyP particles can be purified in a one-step filtration. Finally, abiotic hydrolysis at 95℃ was used to shorten the length of the polyP, allowing the production of homogenous polyP in different lengths. Altogether, this study reports a novel circular P bioeconomy model that successfully valorizes biomass wastes into high-value biomedical materials.

關鍵字(中)

★ 微藻
★ 循環生物經濟
★ 聚磷酸鹽
★ 聚磷酸酶
★ 肌酸
★ 磷酸肌酸
★ 肌酸激酶

關鍵字(英)

★ microalgae
★ circular bioeconomy
★ polyphosphate
★ polyphosphate kinase
★ creatine
★ creatine phosphate
★ creatine kinase

論文目次

摘要................................................................................................................................. i
Abstract .......................................................................................................................... ii
誌謝.............................................................................................................................. iii
目錄............................................................................................................................... iv
圖目錄.......................................................................................................................... vii
表目錄............................................................................................................................ x
符號說明....................................................................................................................... xi
第一章前言............................................................................................................ 1
1.1 研究背景........................................................................................................ 1
1.2 研究動機與目的............................................................................................ 2
第二章文獻回顧.................................................................................................... 3
2.1 磷.................................................................................................................... 3
2.1.1 磷資源之利用與對環境造成的影響............................................ 3
2.1.2 生物除磷技術................................................................................ 4
2.1.3 萃取聚磷酸鹽之技術.................................................................... 8
2.2 酵素.............................................................................................................. 11
2.2.1 酵素動力學.................................................................................. 11
2.2.2 Adenosine kinase（ADK）......................................................... 13
2.2.3 Polyphosphate kinase（PPK） ................................................... 14
2.2.4 Creatine kinase（CK） ............................................................... 16
2.3 產物.............................................................................................................. 16
2.3.1 腺苷三磷酸（Adenosine triphosphate, ATP） .......................... 16
2.3.2 聚磷酸鹽（Polyphosphate, polyP） ........................................... 17 2.3.3 肌酸（Creatine）與磷酸肌酸（Creatine phosphate） ............. 19
第三章材料與方法.............................................................................................. 22
3.1 實驗架構...................................................................................................... 22
3.2 實驗材料與設備.......................................................................................... 23
3.2.1 實驗藥品...................................................................................... 23
3.2.2 實驗設備...................................................................................... 24
3.3 菌種保存及培養.......................................................................................... 26
3.3.1 菌種保存...................................................................................... 26
3.3.2 菌種培養...................................................................................... 26
3.4 蛋白質實驗分析.......................................................................................... 26
v
3.4.1 目標蛋白表達.............................................................................. 27
3.4.2 蛋白質之萃取及純化.................................................................. 27
3.4.3 蛋白質之定性.............................................................................. 28
3.4.4 蛋白質之定量.............................................................................. 29
3.5 豬場廢水採樣點.......................................................................................... 30
3.6 微藻培養...................................................................................................... 30
3.7 聚磷酸鹽實驗分析...................................................................................... 31
3.7.1 聚磷酸鹽之萃取.......................................................................... 31
3.7.2 聚磷酸鹽之定量.......................................................................... 32
3.8 從微藻polyP再生ATP反應 ..................................................................... 32
3.9 利用酵素從微藻polyP中合成磷酸肌酸反應 .......................................... 33
3.10 利用酵素合成長鏈polyP ........................................................................... 33
3.11 將長鏈polyP均質化 .................................................................................. 34
3.11.1 PPX酵素法 ................................................................................. 34
3.11.2 水解法.......................................................................................... 34
3.12 特性分析...................................................................................................... 34
3.12.1 光學顯微鏡.................................................................................. 34
3.12.2 螢光顯微鏡.................................................................................. 34
3.12.3 TBE-Urea gel ............................................................................... 35
3.12.4 高效液相層析.............................................................................. 36
3.12.5 PolyP長度分布的量化 ............................................................... 37
3.12.6 Hexokinase/Glucose‐6‐Phosphate Dehydrogenase定量分析 .... 37
第四章結果與討論.............................................................................................. 38
4.1 微藻聚磷酸鹽.............................................................................................. 38
4.2 利用酵素從微藻polyP中合成磷酸肌酸 .................................................. 40
4.3 利用酵素合成長鏈polyP ........................................................................... 47
4.4 以線性方式催化酵素合成長鏈polyP ....................................................... 50
4.5 將長鏈polyP均質化 .................................................................................. 57
第五章結論與建議.............................................................................................. 61
5.1 結論.............................................................................................................. 61
5.2 建議.............................................................................................................. 61
參考文獻...................................................................................................................... 62
附錄A .......................................................................................................................... 69
vi
從微藻polyP再生ATP .......................................................................................... 69
附錄B .......................................................................................................................... 73

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指導教授

王柏翔(Po-Hsiang Wang)

審核日期

2023-6-20

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