幾丁聚醣經化學或酵素降解後的主要產物為低分子量幾丁聚醣(low molecular weight chitosan)和幾丁寡醣(chitooligosaccharide)，本論文分別以毛細管區帶電泳(capillary zone electrophoresis)和毛細管凝膠電泳(capillary gel electrophoresis)分析低分子量幾丁聚醣樣品的去乙醯化程度(degree of deacetylation, DDA)和分子量。而幾丁寡醣樣品的組成分析則以基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF/MS)進行，並根據所得質譜訊號計算樣品平均去乙醯化程度。本實驗所開發的毛細管電泳和MALDI-TOF/MS分析方法具有快速檢測、高效率和微量分析的特性，有利於我們從幾丁聚醣降解的一鍋反應中(one pot reaction)，探討在不同反應條件下的幾丁聚醣降解反應性時程。
利用過硫酸鈉(sodium persulfate, NaPS)降解幾丁聚醣(DDA85%)所得低分子量幾丁聚醣和幾丁寡醣產物之DDA與原始樣品相近；但利用胃蛋白酶(pepsin)降解相同樣品經反應72小時後卻得到DDA90%的低分子量幾丁聚醣和DDA58%的幾丁寡醣兩種不同DDA產物。以MALDI-TOF/MS分析兩種降解反應在3小時所得幾丁寡醣樣品的組成，我們發現利用酵素所切出的寡醣片段大多以低DDA產物為主，而利用NaPS所切出的樣品則是具高DDA的片段較多，據此結果推論，酵素對於幾丁聚醣長鏈會有選擇性地針對低DDA部分進行水解反應，而NaPS則能在鏈上兩種單體組成間進行均勻降解。為了探討樣品DDA對上述結論的影響，我們將DDA85%的樣品進行不同程度的乙醯化反應，得到兩種不同去乙醯化程度(DDA55%和33%)但分子量相近的幾丁聚醣樣品，並分別以NaPS和胃蛋白酶、脂肪水解酶(lipase A)、纖維素酶(cellulase)、鳳梨酵素(bromelain)以及木瓜酵素(papain)等五種酵素，對三種DDA幾丁聚醣樣品進行降解反應。實驗結果顯示由NaPS降解所得低分子量幾丁聚醣和幾丁寡醣產物的DDA皆與原始樣品相同；而以酵素降解時則會因所使用酵素性質和幾丁聚醣DDA不同而得到具不同DDA的降解產物。
   另外，我們也探討了完全去乙醯化幾丁寡醣(DDA100%)分別在酸性、中性和鹼性條件下的乙醯化反應性差異，並因此得到完全乙醯化幾丁寡醣(DDA0%)的最佳反應條件。在以MALDI-TOF/MS分析兩種相同濃度的樣品時，我們發現DDA0%樣品之MS訊號大小是DDA100%樣品的4~6倍。

The major products of chitosan degraded by chemical or enzymes are low molecular weight chitosan (low Mw chitosan) and chitooligosaccharide. In this thesis we utilized capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE) to analyze the degree of deacetylation (DDA) and the molecular weight of the low Mw weight chitosan product. Furthermore, the composition analysis of chitooligosaccharide product was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF/MS), and the averaged DDA was calculated based on the relative signals of chitooligosaccharides from mass spectra. Our CE and MALDI-TOF/MS methods feature fast measurement, high throughput, and microanalysis, and thus are advantageous to the time course study of various degradation reactions of chitosan from the one pot reaction.
    The DDA’s of low Mw chitosan and chitooligosaccharide produced from chitosan degradation by sodium persulfate (NaPS) are close to the DDA of the original chitosan sample (DDA85%). However, low Mw chitosan with DDA90% and chitooligosaccharide with DDA58% were obtained if pepsin was used to digest the same chitosan for 72 hours. Analyzing the compositions of the chitooligosaccharide products for both reactions after 3-hour degradation by MALDI-TOF/MS, we found that the major products obtained from pepsin digestion are chitooligosaccharides with low DDA’s. On the other hand, the NaPS degradation products are mainly chitooligosaccharides with high DDA’s. According to the results we infer that enzyme can selectively hydrolyze chitosan polymer chains on the low DDA regions, but NaPS seems to homogeneously fragment the polymer chains regardless of the monomer compositions. In order to study the effects of sample DDA on the aforementioned conclusion, we carried out different degrees of acetylation reactions to convert the DDA85% sample into two chitosan samples with similar molecular weight but different DDA’s at 55% and 33%, respectively. The three chitosan samples were then separately subjected to degradation reactions with NaPS and enzymes such as pepsin, lipase A, cellulase, bromelain, and papain. The experimental results revealed that the DDA’s of the low Mw chitosan and chitooligosaccharide produced by the NaPS degradation are the same as the original chitosan samples, while the DDA’s of the enzymatic digestion products are influenced by the type of enzymes as well as the DDA’s of the chitosan substrates.
    Additionally, we also investigated the differences in acetylation reactivity of fully deacetylated chitooligosaccharide (DDA100%) sample under acidic, neutral, and basic reaction conditions, and consequently obtained the optimal acetylation conditions to generate the fully acetylated chitooligosaccharide (DDA0%) sample. When analyzing the two chitooligosaccharide samples with the same concentration by MALD-TOF/MS, we found that the DDA0% samples are 4 to 6 times the MS signals of the DDA100% samples.

目錄
第一章 緒論...............................................1
1.1前言..................................................1
1.2研究動機...............................................2
1.3幾丁質、幾丁聚醣簡介.....................................3
1.4酵素介紹...............................................4
1.4.1鳳梨酵素(Bromelain)..................................4
1.4.2纖維素酶(Cellulase)...................................5
1.4.3脂肪酶(Lipase A).....................................5
1.4.4木瓜蛋白酶(Papain)....................................6
1.4.5胃蛋白酶(Pepsin).....................................6
1.5幾丁聚醣之降解反應.......................................7
1.6毛細管電泳簡介..........................................8
1.6.1毛細管電泳介紹........................................8
1.6.2毛細管電泳原理........................................8
1.6.3電泳遷移率(electrophoretic mobility).................8
1.6.4電泳滲透流(electroosmotic flow,EOF)..................9
1.6.5毛細管電泳的分離方法..................................11
1.6.6樣品注入方式........................................12
1.7 MALDI-TOF/MS簡介....................................13
1.8測量幾丁聚醣降解產物之去乙醯化程度與分子量..................16
1.8.1利用毛細管電泳分析幾丁聚醣.............................16
1.8.2利用MALDI-TOF/MS 測量幾丁寡醣之乙醯化程度...............18
1.8.3利用核磁共振法測量幾丁聚醣之去乙醯化程度..................18
1.8.4利用黏度計測量幾丁聚醣的極限黏度分子量....................20
1.9本章參考文獻...........................................21
第二章 實驗...............................................28
2.1實驗藥品與器材..........................................28
2.2實驗儀器..............................................32
2.3步驟與方法.............................................35
2.3.1毛細管處理...........................................35
2.3.2毛細管電泳相關實驗的緩衝溶液之配置........................35
2.3.3以毛細管電泳測量去幾丁聚醣DDA之條件和步驟.................36
2.3.4以毛細管電泳測量幾丁聚醣分子量之條件和步驟.................38
2.3.5以毛細管電泳測量分子量關係之條件和步驟.....................39
2.3.6利用核磁共振法測量幾丁聚醣之去乙醯化程度...................44
2.3.7利用MALDI-TOF/MS分析幾丁寡醣樣品.......................45
2.3.8利用ATR FT-IR分析幾丁聚醣樣品..........................48
2.4實驗樣品備製............................................49
2.4.1幾丁聚醣與幾丁寡醣之乙醯化反應...........................49
2.4.2幾丁聚醣之降解反應.....................................51
2.4.3以Silica Gel純化幾丁寡醣中的氯離子......................53
2.5本章參考文獻............................................54

第三章 結果與討論...........................................55
3.1幾丁寡醣之乙醯化反應......................................55
3.1.1醋酸酐濃度對於幾丁寡醣乙醯化反應性之影響...................55
3.1.2溶劑對於幾丁寡醣乙醯化反應性之影響........................59
3.1.3鹼性條件下醋酸酐濃度對於幾丁寡醣乙醯化反應性之影響...........68
3.1.4利用Silica Gel純化幾丁寡醣再進行乙醯化反應................68
3.2完全乙醯化(DDA 0%)與完全去乙醯化(DDA 100%)幾丁寡醣MALDI MS訊號大小之比較..................................................73
3.3以pepsin降解不同DDA幾丁聚醣..............................76
3.3.1以pepsin降解不同DDA幾丁聚醣所得低分子量幾丁聚醣產物之比較....76
3.3.2以pepsin降解不同DDA幾丁聚醣所得幾丁寡醣產物之比較..........89
3.3.3以pepsin降解不同DDA幾丁聚醣所得幾丁寡醣與低分子量幾丁聚醣產物之比較.......................................................95
3.4分別以Sodium persulfate和Pepsin降解不同DDA幾丁聚醣所得產物之比較	........................................................100
3.5分別以不同酵素降解不同去乙醯化程度的幾丁聚醣.................114
3.6本章參考文獻...........................................137
第四章 結論...............................................138

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第二章
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第三章
3.6本章參考文獻
1.	Ravindra, R.; Krovvidi, K. R.; Khan, A. A., “ Solubility parameter of chitin and chitosan”, Carbohydrate Polymers, 1998, 36, 121-127.
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