於鋁基金屬有機骨架材料中封裝蔗糖分解酶 與其串聯生物催化反應之研究

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盧昱丞(Yu-Cheng Lu) 查詢紙本館藏

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化學學系

論文名稱

於鋁基金屬有機骨架材料中封裝蔗糖分解酶與其串聯生物催化反應之研究
(Encapsulation of Invertase in Aluminum-Based Metal-organic Frameworks for Tandem Biocatalysis)

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至系統瀏覽論文 (2029-8-20以後開放)

摘要(中)

本研究旨在探討蔗糖分解酶 (Invertase, INV) 酵素固定化於鋁金屬基金屬有機骨架材料 (A520 和 Al-MIL-53) 上的應用，以優化轉化糖漿的生產。蔗糖分解酶能將蔗糖水解為葡萄糖和果糖，是製備轉化糖漿的重要催化劑。轉化糖漿 (Invert syrup) 因其較高的甜度和良好的保濕性，在食品工業中具有廣泛的應用，例如在烘焙和飲料行業中被廣泛使用。鋁金屬基金屬有機骨架材料 (Metal-organic Frameworks, MOFs)，如 A520 和 Al-MIL-53 ，因其高比表面積、可調控的孔徑和優異的穩定性，成為固定化酵素的理想載體。
本研究結果發現蔗糖分解酶固定化於 A520 和 Al-MIL-53 中，能顯著提高酵素的穩定性和活性。例如在反應溫度為 55°C 和 65°C 的高溫條件下將蔗糖分解酶包覆於 A520 材料中，其活性均較純蔗糖分解酶高出約 2 至 4 倍。這些 MOFs 材料能夠保護酵素在不利的反應條件（如高溫或強酸強鹼環境）下仍能保持其催化功能，從而提高反應效率和產品品質。此外，這種固定化方法還能有效防止酵素的溢漏問題，實現酵素的重複利用，降低生產成本。
未來的研究方向包括將酵素串聯反應（Tandem reactions）應用於固定化系統。例如，將 α-半乳糖苷酶（α-Galactosidase）和蔗糖分解酶聯合固定於 A520 和 Al-MIL-53 中，可實現棉子糖（Raffinose）一步轉化為高價值的轉化糖漿。此方法不僅簡化生產工藝，減少中間產物分離和酵素再利用步驟，還顯著提高生產效率和經濟效益。
綜上所述，本研究展示了 MOFs 在酵素固定化中的巨大潛力，為食品工業中的轉化糖漿生產提供高效且經濟的技術途徑。同時，這一技術在酵素串聯反應中的應用前景廣闊，有望實現多步驟反應的一體化操作，進一步提升工業生產效率和永續性。

摘要(英)

This study aims to investigate the application of invertase (INV) enzyme immobilization on aluminum-based metal-organic frameworks (MOFs), specifically A520 and Al-MIL-53, to optimize the production of invert syrup. Invertase catalyzes the hydrolysis of sucrose into glucose and fructose, serving as a crucial catalyst in the preparation of invert syrup. Due to its higher sweetness and excellent moisture retention properties, invert syrup is widely used in the food industry, particularly in baking and beverages. Aluminum-based MOFs, such as A520 and Al-MIL-53, are ideal carriers for enzyme immobilization due to their high specific surface area, tunable pore size, and exceptional stability.
The results of this study indicate that immobilizing invertase on A520 and Al-MIL-53 significantly enhances enzyme stability and activity. For instance, under high-temperature reaction conditions of 55°C and 65°C, invertase encapsulated in A520 material exhibited an activity approximately 2 to 4 times higher than that of pure invertase. These MOFs can protect the enzyme′s catalytic function under adverse reaction conditions, such as high temperatures or strong acidic and alkaline environments, thereby improving reaction efficiency and product quality. Additionally, this immobilization method effectively prevents enzyme leakage, enabling enzyme reuse and reducing production costs.
Future research directions include applying tandem reactions in immobilized systems. For example, co-immobilizing α-galactosidase and invertase on A520 and Al-MIL-53 could achieve the one-step conversion of raffinose to high-value invert syrup. This approach simplifies the production process, reduces intermediate product separation and enzyme reuse steps, and significantly enhances production efficiency and economic benefits.
In summary, this study demonstrates the significant potential of MOFs in enzyme immobilization, providing an efficient and economical technological approach for the production of invert syrup in the food industry. Moreover, this technology has broad application prospects in tandem enzyme reactions, potentially enabling the integration of multi-step reactions and further enhancing industrial production efficiency and sustainability.

關鍵字(中)

★ 鋁基底金屬骨架有機材料
★ 蔗糖轉換酶
★ 酵素固定化
★ 溫和水相合成法
★ 轉化糖漿

關鍵字(英)

★ aluminum-based metal-organic frameworks
★ invertase
★ enzyme immobilization
★ mild aqueous synthesis method
★ invert syrup

論文目次

中文摘要 i
Abstract iii
目錄 v
圖目錄 ix
表目錄 xii
第一章緒論 1
1-1 金屬有機骨架材料 1
1-2 鋁金屬基底之金屬有機骨架材料 3
1-2-1 金屬有機骨架材料 A520 4
1-2-2 金屬有機骨架材料 Al-MIL-53 5
1-3 酵素、酵素固定化與金屬有機骨架材料 7
1-4 酵素串聯反應 (Tandem Reactions) 9
1-5 蔗糖分解酶 (Invertase, INV) 10
1-6 棉子糖 (Raffinose) 10
1-7 研究動機與目的 11
第二章實驗部分 13
2-1 實驗藥品 13
2-2 實驗使用儀器 16
2-3 實驗鑑定用儀器 17
2-4 實驗儀器之原理 18
2-4-1 紫外光可見光分光光譜儀 (UV/VIS Spectrophotometer) 18
2-4-2 X 射線粉末繞射儀 (Powder X-ray Diffractometer；XRD) 20
2-5 實驗所用之分析方法 25
2-5-1 Bradford Assay: 偵測蛋白質濃度 25
2-5-2 糖脎反應:用於測定蔗糖分解酶活性 26
2-5-3 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (SDS-PAGE) 27
2-6 實驗步驟 29
2-6-1 鋁金屬基底金屬有機材料 A520 之合成 29
2-6-2 蔗糖分解酶封裝於鋁金屬基底金屬有機材料 A520 (INV@A520) 之合成 29
2-6-3 鋁金屬基底金屬有機材料 Al-MIL-53 之合成 30
2-6-4 蔗糖分解酶封裝於鋁金屬基底金屬有機材料 Al-MIL-53 (INV@MIL-53) 之合成 30
2-6-5 測定蛋白質含量 (Bradford Assay) 31
2-6-6 配製糖脎反應試劑與檢量線 32
2-6-7 測定蔗糖分解酶活性 33
2-6-8 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE) 35
第三章結果與討論 37
3-1 蔗糖分解酶包覆於 A520 之鑑定與活性實驗 37
3-1-1 INV@A520 之 X 射線粉末繞射圖譜鑑定 37
3-1-2 INV@A520之掃描式電子顯微鏡影像分析 38
3-1-3 INV@A520 之 SDS-PAGE 鑑定 39
3-1-4 INV@A520 之蛋白質含量測試 40
3-1-5 INV@A520 與純蔗糖分解酶之活性探討 41
3-2 蔗糖分解酶包覆於 Al-MIL-53 之鑑定與活性實驗 49
3-2-1 INV@MIL-53 之 X 射線粉末繞射圖譜鑑定 49
3-2-2 INV@MIL-53 之掃描式電子顯微鏡影像分析 50
3-2-3 INV@MIL-53 之 SDS-PAGE 鑑定 52
3-2-4 INV@MIL-53之蛋白質含量測試 53
3-2-5 蔗糖分解酶包覆於 A520 與 Al-MIL-53 和純蔗糖分解酶的活性實驗結果比較 54
第四章結論及未來展望 60
參考文獻 62

圖目錄
圖 1 1 各種合成 MOFs 的方法12 2
圖 1-2 MOFs 之應用 2
圖 1-3 A520 之結構示意圖 5
圖 1 5 固定化酵素之方法53, 59 8
圖 1 6 酵素-MOF複合材料之製備方法65 8
圖 1 7 棉子糖透過雙酵素反應產生轉化糖漿之示意圖 12
圖 2-1 電磁波能量範圍79 18
圖 2-2 雙光紫外光－可見光光譜儀結構示意圖81 19
圖 2 3 晶面方向與立方晶體中不同晶面的密勒指數82, 83 20
圖 2 4 簡單立方點陣中不同晶面的間距82, 83 21
圖 2 5 布拉格定律示意圖83 22
圖 2-6 掃描式電子顯微鏡結構示意圖86 24
圖 2-7 糖脎反應 26
圖 2-8 SDS-PAGE 凝膠電泳示意圖90 28
圖 2-9 本次研究所使用的 Protein Marker 28
圖 3-1 INV@A520之 X 射線粉末繞射圖譜 37
圖 3-2 INV@A520 之掃描式電子顯微鏡影像分析圖 38
圖 3-3 INV@A520 之 SDS-PAGE 鑑定圖 39
圖 3-4 INV@A520 之 Bradford Assay 檢量線 40
圖 3-5 INV@A520 與 Free Invertase 在 Incubation 2 分鐘後分解蔗糖之活性測試 (反應條件：pH 4.0, 反應溫度 55°C) (三重複) 41
圖 3-6 INV@A520 與 Free Invertase 在 Incubation 5 分鐘後分解蔗糖之活性測試 (反應條件：pH 4.0, 反應溫度 55°C) (三重複) 42
圖 3-7 INV@A520 與 Free Invertase 在 Incubation 10 分鐘後分解蔗糖之活性測試 (反應條件：pH 4.0, 反應溫度 55°C) (三重複) 42
圖 3-8 INV@A520 與 Free Invertase 在 Incubation 20 分鐘後分解蔗糖之活性測試 (反應條件：pH 4.0, 反應溫度 55°C) (三重複) 43
圖 3-9 INV@A520與 Free Invertase 在 pH 4.0 且反應溫度為 45°C 下分解蔗糖之活性測試比較圖 (三重複) 44
圖 3-10 INV@A520與 Free Invertase 在 pH 4.0 且反應溫度為 55°C 下分解蔗糖之活性測試比較圖 (三重複) 44
圖 3-11 INV@A520與 Free Invertase 在 pH 4.0 且反應溫度為 65°C 下分解蔗糖之活性測試比較圖 (三重複) 45
圖 3-12 INV@A520與 Free Invertase 在 pH 5.0 且反應溫度為 45°C 下分解蔗糖之活性測試比較圖 (三重複) 45
圖 3-13 INV@A520與 Free Invertase 在 pH 5.0 且反應溫度為 55°C 下分解蔗糖之活性測試比較圖 (三重複) 46
圖 3-14 INV@A520與 Free Invertase 在 pH 5.0 且反應溫度為 65°C 下分解蔗糖之活性測試比較圖 (三重複) 46
圖 3-15 INV@A520與 Free Invertase 在 pH 6.0 且反應溫度為 45°C 下分解蔗糖之活性測試比較圖 (三重複) 47
圖 3-16 INV@A520與 Free Invertase 在 pH 6.0 且反應溫度為 55°C 下分解蔗糖之活性測試比較圖 (三重複) 47
圖 3-17 INV@A520與 Free Invertase 在 pH 6.0 且反應溫度為 65°C 下分解蔗糖之活性測試比較圖 (三重複) 48
圖 3-18 INV@MIL-53 與 Al-MIL-53 之 X 射線粉末繞射圖譜 49
圖 3-19 INV@MIL-53 之掃描式電子顯微鏡影像分析圖 50
圖 3-20 INV@MIL-53 之 SDS-PAGE 鑑定圖 52
圖 3-21 INV@MIL-53 之 Bradford Assay 檢量線 53
圖 3-22 INV@A520、INV@MIL-53 與 Free Invertase 在 pH 4.0且反應溫度為 45°C下分解蔗糖之活性測試 (三重複) 54
圖 3-23 INV@A520、INV@MIL-53 與 Free Invertase 在 pH 4.0且反應溫度為 55°C下分解蔗糖之活性測試 (三重複) 55
圖 3-24 INV@A520、INV@MIL-53 與 Free Invertase 在 pH 4.0且反應溫度為 65°C下分解蔗糖之活性測試 (三重複) 55
圖 3-25 INV@A520、INV@MIL-53 與 Free Invertase 在 pH 5.0且反應溫度為 45°C下分解蔗糖之活性測試 (三重複) 56
圖 3-26 INV@A520、INV@MIL-53 與 Free Invertase 在 pH 5.0且反應溫度為 55°C下分解蔗糖之活性測試 (三重複) 56
圖 3-27 INV@A520、INV@MIL-53 與 Free Invertase 在 pH 5.0且反應溫度為 65°C下分解蔗糖之活性測試 (三重複) 57
圖 3-28 INV@A520、INV@MIL-53 與 Free Invertase 在 pH 6.0且反應溫度為 45°C下分解蔗糖之活性測試 (三重複) 57
圖 3-29 INV@A520、INV@MIL-53 與 Free Invertase 在 pH 6.0且反應溫度為 55°C下分解蔗糖之活性測試 (三重複) 58
圖 3-30 INV@A520、INV@MIL-53 與 Free Invertase 在 pH 6.0且反應溫度為 65°C下分解蔗糖之活性測試 (三重複) 58
圖 3-31 Free Invertase、 INV@A520 與 INV@MIL-53 於不同溫度、酸鹼值下之活性比較示意圖 59

表目錄

表 1 實驗藥品 13
表 2 分離/膠集膠體配方 35
表 3 染劑/退染劑配方 36

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指導教授

謝發坤(Fa-Kuen Shieh)

審核日期

2024-8-21

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