果膠酯酶( pectinesterase, PE )會水解果膠上半乳糖醛酸之甲氧基，釋放出甲醇，進而降低果膠的酯化度。近年來有研究指出植物來源之PE於進行水解反應的同時，亦催化轉醯化反應，而微生物來源之PE則未見文獻探討，故本研究以微生物(Aspergillus niger)生產之商業果膠酵素的PE，證實此PE是否具催化轉醯化反應之能力，以及探討其催化轉醯化的最適條件。研究結果顯示，去酯化反應的最適條件，係在無氯化鈉的存在下，以pH 5.0在40℃下反應，有最高的活性表現。採用雷射粒徑分析法之方式，可證實Aspergillus niger PE會催化轉醯化反應，在果膠分子間產生新酯鍵，使果膠的分子量變大。轉醯化反應的程度與環境因子有關，在0.3%濃度下之果膠較適於 Aspergillus niger PE催化轉醯化反應，而PE酵素活性或反應時間的增加，亦會使果膠分子的粒徑明顯增加，其最適反應條件為pH 3.0~3.35，溫度為40℃。根據實驗結果顯示，Aspergillus niger 之PE所催化的轉醯化反應與其活性並非呈正相關。此外，Aspergillus niger 之PE所催化的轉醯化反應，在pH 3.35及3.0下反應有較大的粒徑變化， 因此pH值在轉醯化反應系統扮演相當重要的角色。

Pectinesterase (PE) will hydrolyze the methoxyl groups in the galacturonic acids of pectin to release methanol and the degree of esterification will be decreasing. Recent researches indicated that transacylation reaction occurred simultaneously when the de-esterification reaction progressed by PE through the analysis of gel permeation chromatography, viscometer and Instron etc.. The PE catalyzed the transacylation, and the new forms of ester linkages between pectin molecules also will be illustrated, and the increscent in the molecular weight of pectin in the presence of PE during incubation. Results showed that PE from Aspergillus niger remarkably conducted the transacylation that was affected by some factors, the optimal conditions for transacylation by Aspergillus niger PE were 40 ℃ and at pH 3.0~3.35 by laser particle size analysis. The transacylation catalyzed by PE from Aspergillus niger is not showed positive relation to it’s enzyme activity, the range of particle size is wide in pH 3.35 and 3.0, the pH value plays an important role in transacylation reaction.

中文摘要Ⅰ
AbstractⅡ
謝誌Ⅲ
目錄Ⅳ
圖表目錄Ⅸ
1、前言1
2、文獻回顧2
2.1果膠源起2
2.2果膠結構2
2.3商業果膠2
2.3.1商業果膠之來源7
2.3.2商業果膠之萃取7
2.3.3高酯化度果膠之製造方式7
2.3.4低酯化度果膠之製造方式7
2.4果膠的物理性質9
2.4.1粉末性質9
2.4.2分子量9
2.4.3儲存安定性9
2.4.4黏度9
2.4.5溶解度10
2.4.6凝膠性10
2.4.6.1高甲氧基果膠之凝膠原理10
2.4.6.2低甲氧基果膠之凝膠原理13
2.5果膠的化學性質13
2.5.1果膠安定13
2.5.2果膠酯化度14
2.5.3果膠分子的去酯化作用14
2.5.4果膠分子的裂解17
2.5.4.1β-脫去反應17
2.5.4.2水解反應17
2.6果膠在食品上之應用18
2.7市售果膠酵素20
2.8果膠酯酶20
2.8.1果膠酯酶之來源20
2.8.1.1植物性來源20
2.8.1.2微生物來源21
2.8.2果膠酯酶之化學組成21
2.8.3果膠酯酶之作用方式25
2.8.4果膠酯酶之同功酶作用25
2.8.5果膠酯酶去酯化活性測定25
2.8.5.1鹼滴定法25
2.8.5.2比色法27
2.8.6影響果膠酯酶活性之因素27
2.8.6.1溫度27
2.8.6.2酸鹼值27
2.8.6.3金屬離子之種類及濃度29
2.9果膠酵素分離純化29
2.9.1粗酵素液的製備29
2.9.2硫酸銨鹽析29
2.9.3層析法30
2.9.4有機溶劑沉澱法31
2.9.5其它分離純化之分析方法31
2.10果膠酯酶所催化轉醯化反應31
2.10.1轉醯化反應可能之機制31
2.10.2轉醯化反應結果之檢測34
2.10.2.1膠濾層析法34
2.10.2.2膠體硬度34
2.10.2.3黏度36
2.10.2.4混濁度36
2.10.2.5雷射粒徑分析儀36
3、材料與方法37
3.1實驗材料37
3.1.1豌豆莢皮37
3.1.2商業果膠酵素37
3.2儀器設備37
3.3化學試藥38
3.4實驗架構40
3.5植物組織酒精不溶物交聯處理及提高酯化度之處理40
3.5.1植物組織酒精不溶物之製備40
3.5.2植物組織交聯產物之製備40
3.5.3高酯化度植物組織酒精不溶物之製備40
3.6以植物組織管柱層析純化果膠酵素試驗42
3.6.1高酯化度植物組織酒精不溶物管柱42
3.7商業果膠分解酵素之純化及純度分析42
3.7.1高酯化度交聯化酒精不溶物膠體層析法42
3.7.2膠濾層析法42
3.7.3 SDS不連續聚丙烯醯胺膠體電泳製備43
3.8酵素活性測定47
3.8.1果膠酯酶活性測定47
3.8.1.1鹼滴定法47
3.8.1.2比色法47
3.8.2聚半乳醣醛酸酶測定48
3.8.3DNS指示劑之配製流程48
3.8.4果膠分解酵素活性測定48
3.9蛋白質含量測定49
3.10.1最適pH值49
3.10.2最適溫度49
3.10.3溫度耐受性49
3.10.4最適鹽濃度49
3.11商業果膠酵素之PE催化轉醯化反應條件之探討50
3.11.1果膠受質之前處理50
3.11.2轉醯化反應條件之探討50
3.11.2.1果膠濃度50
3.11.2.2 pH值50
3.11.2.3酵素活性50
3.11.2.4鹽濃度51
3.11.2.5反應時間51
3.11.2.6溫度51
3.11.2.7果膠酯酶溫度耐受性51
3.11.2.8添加氯化鈣對轉醯化反應之影響52
3.11.3雷射粒徑分析儀檢測轉醯化反應52
4、結果與討論53
4.1市售果膠酵素之PE純化53
4.1.1交聯化酒精不溶物親和性管柱層析53
4.1.2超過濾濃縮56
4.1.3膠濾層析56
4.2電泳分析56
4.3 Aspergillus niger PE之最適去酯化活性探討59
4.3.1酸鹼值59
4.3.2溫度59
4.3.3熱穩定性64
4.3.4鹽濃度64
4.3.5氯化鈣之添加64
4.4 Aspergillus niger PE所催化轉醯化反應之探討65
4.4.1以雷射粒徑分析儀偵測果膠分子粒徑變化68
4.4.2果膠濃度對轉醯化反應之影響68
4.4.3 PE活性對轉醯化反應之影響69
4.4.4 pH對轉醯化反應之影響69
4.4.5溫度對轉醯化反應之影響73
4.4.5.1最適溫度73
4.4.5.2熱穩定性試驗73
4.4.6反應時間對轉醯化反應之影響76
4.5 PE去酯化活性與其所催化轉醯化反應結果之比較80
5、結論81
參考文獻82
作者簡介93

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