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研究生: 王楷澄
WANG, KAI-CHENG
論文名稱: 以一鍋式酵素合成法將蔗渣木糖與菸鹼醯胺轉化為菸鹼醯胺腺嘌呤二核苷酸
One-pot enzymatic synthesis of NADH from bagasse-derived xylose and nicotinamide
指導教授: 林進榮

王柏翔
口試委員:
學位類別: 碩士
Master
系所名稱: 工學院 - 環境工程研究所
Graduate Institute of Environmental Engineering
論文出版年: 2025
畢業學年度: 113
語文別: 中文
論文頁數: 65
中文關鍵詞: 甘蔗渣木糖廢棄物加值一鍋式酵素級聯綠色經濟
外文關鍵詞: Bagasse, Xylose, Waste valorization, One-pot enzymatic cascade, Green economy
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  • 甘蔗渣(bagasse)是最常見且產量最大的纖維素類農業副產物之一,為糖業
    壓榨後剩餘的木質纖維性殘渣。儘管多數甘蔗渣僅作為鍋爐燃料使用,但其豐富
    的半纖維素含量使其成為極具潛力的可再生碳源。本研究以永續發展目標第 12
    項「責任消費與生產」為指引,建立一套將甘蔗渣資源化的廢棄物加值利用系統。
    透過酸水解處理甘蔗渣以回收 D-木糖,進一步結合菸鹼醯胺(NAM)進行酵素催
    化反應,實現一鍋式酵素級聯合成菸鹼醯胺腺嘌呤二核苷酸(NADH)。
    本系統整合11種經重組表現酵素,並結合聚磷酸鹽驅動之ATP再生機制,
    有效推進反應進行,同時避免中間產物純化與多餘廢棄物產生。最終NADH濃
    度可達2.7 mM(~1.8 g/L),轉化效率高達85%。此外,唯一副產物為正磷酸鹽,
    可由微藻轉化為polyP進行循環利用,展示本系統良好的綠色化學與資源循環潛
    力。
    綜合而言,本研究展示了一個以農業廢棄物甘蔗渣為原料、具有經濟效益與
    環保潛力的NADH生產平台,為推動綠色經濟與永續生產提供具體實證與應用
    潛力。


    Bagasse is one of the most common and abundant cellulose-based agricultural
    residues, representing the fibrous lignocellulosic byproduct remaining after sugarcane
    juice extraction. Although bagasse is mostly used as boiler fuel, its rich hemicellulose
    content makes it a promising renewable carbon source. In alignment with Sustainable
    Development Goal 12: Responsible Consumption and Production, this study establishes
    the waste valorization platform to convert bagasse into valuable biochemicals. Through
    acid hydrolysis, D-xylose was recovered from bagasse, and was subsequently reacted
    with nicotinamide (NAM) via an enzyme-catalyzed process to achieve one-pot cascade
    synthesis of nicotinamide adenine dinucleotide (NADH).
    The system integrates 11 recombinant enzymes and incorporates a polyphosphate
    driven ATP regeneration mechanism, enabling efficient NADH biosynthesis while
    avoiding intermediate purification and excess waste generation. The final NADH
    concentration reached 2.7 mM (~1.8 g/L) with a conversion efficiency of up to 85%.
    Furthermore, the only byproduct, inorganic phosphate, can be recycled by microalgae
    into polyphosphate (polyP), demonstrating the system’s potential in green chemistry
    and resource circularity.
    In summary, this study presents an environmentally and economically viable
    NADH production platform using agricultural waste bagasse, offering a practical
    solution for promoting green economy and sustainable production.

    目錄 摘要 i Abstract ii 致謝 iii 目錄 iv 圖目錄 vi 表目錄 viii 第一章 前言 1 1.1 研究背景 1 1.2 研究動機與目的 3 1.3 實驗架構 4 第二章 文獻回顧 6 2.1 D-木糖 6 2.2 酵素 6 2.2.1 酵素動力學 7 2.2.2 木糖異構酶 (XI) 9 2.2.3 木酮糖激酶 (XK) 10 2.2.4 磷酸核酮糖-3-差向異構酶 (RPE) 11 2.2.5 核糖-5-磷酸異構酶 (RPI) 12 2.2.6 磷酸核糖焦磷酸合成酶 (PRPS) 12 2.2.7 菸鹼醯胺磷酸核糖轉移酶 (NAMPT) 13 2.2.8 無機焦磷酸酶 (PPase) 14 2.2.9 多磷酸激酶 (PPK2) 14 2.2.10 菸鹼醯胺單核苷酸腺苷酸轉移酶 (NMNAT) 15 2.2.11 磷酸鹽脫氫酶 (PTDH) 15 2.3 菸鹼醯胺單核苷酸 (NMN) 16 2.4 菸鹼醯胺腺嘌呤二核苷酸 (NADH) 16 第三章 材料與方法 18 3.1 實驗架構 18 3.2 實驗材料與設備 19 3.2.1 實驗藥品 19 3.2.2 實驗設備 21 3.3菌種培養與保存 22 3.3.1 菌種保存 22 3.3.2 菌種培養 22 3.4 目標蛋白表達 22 3.5 蛋白質表達與純化 23 3.6 蛋白質之定性 25 3.7 蛋白質之定量 26 3.8 甘蔗渣衍生木糖酸水解前處理與定量分析 26 3.9 一鍋式酵素法合成Xu5P 27 3.10一鍋式酵素法合成NADH 28 3.11聚磷酸鹽 (polyphosphate)之檢測 28 3.12 NADH之檢測 29 3.13高效液相層析 (High Performance Liquid Chromatography, HPLC) 29 第四章 結果與討論 31 4.1 甘蔗渣酸水解後定量分析 31 4.2 一鍋式酵素法合成Xu5P 32 4.3 一鍋式酵素法合成NADH 34 4.3.1 未優化前合成NADH 34 4.3.2 改變木糖濃度對一鍋式酵素合成NADH之影響 36 4.3.3 改變NAM濃度對一鍋式酵素合成NADH之影響 38 4.3.4 改變ATP濃度對一鍋式酵素合成NADH之影響 39 4.3.5 改變反應pH對一鍋式酵素合成NADH之影響 40 4.3.6 反應緩衝溶液對一鍋酵素合成NADH之影響 41 第五章 結論與建議 44 5.1 結論 44 5.2 建議 44 參考文獻 46 附錄 52

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