本論文主要是以毛細管電泳(capillary electrophoresis)和基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF/MS)技術研究幾丁質和幾丁聚醣的三種衍生化反應，包括去乙醯化反應、乙醯化反應、和酵素降解反應。CE可偵測樣品的平均去乙醯化程度(degree of deacetylation, DDA)及其分佈情形，而 MALDI-TOF/MS則可分析幾丁聚醣產物(chitooligomers)的單體組成及平均DDA大小。利用去乙醯化反應和再乙醯化反應，可以將DDA分佈寬廣且不對稱的幾丁聚醣樣品改質成為DDA分佈窄且對稱的樣品。由幾丁質去乙醯化動態反應的實驗結果顯示，多階段去乙醯化反應不但能增加反應效率，且能備製具高DDA的幾丁聚醣樣品。此外，幾丁聚醣經胃蛋白酶(pepsin)降解後的主要產物為低分子量幾丁聚醣(LMwtC)和幾丁寡糖(chitooligomers)，在鹼性條件下加入適當的甲醇，可有效分離此兩種產物，並分別以CE和MALDI-TOF/MS進行分析。根據隨著反應時間增加所測得兩種產物的產率、平均DDA、和DDA分佈之變化情形顯示，pepsin降解幾丁聚醣的反應效率隨著原始樣品的平均DDA和分子量降低而增加，而且我們發現pepsin有趨向於降解幾丁聚醣長鏈上富含乙醯基區域的現象。

In this thesis capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) were employed to study three derivation reactions for chitin and chitosan, including deacetylation, reacetylation, and enzymatic degradation reactions. CE was used to determine the average degree of deacetylation (DDA) and DDA distribution of chitosan sample, while MALDI-TOF/MS was utilized to analyze the monomer compositions and average DDA of chitooligomers. The broad and asymmetric DDA distribution of chitosan sample can be modified to become narrow and symmetric by performing deacetylation and reacetylation reactions. According to the results obtained from the time course deacetylation of chitin, multi-stage deacetylation reaction not only can increase the reaction efficiency but also can produce chitosan with high DDA. In addition, the major products of chitosan degradation by pepsin digest are low molecular weight chitosan (LMwtC) and chitooligomers, which can be separated under basic condition with appropriate methanol concentration and then are subject to CE and MS analysis. Based on the variations of product yields, average DDAs, and DDA distributions measured with increasing reaction time, we found that the efficiency of chitosan degradation by pepsin digest would increase with decreasing average DDA and molecular weight of the chitosan samples. Moreover, pepsin would prefer to cleave the polysaccharide chain on the acetyl group rich domains.

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毛細管電泳簡介	5
1.4.1毛細管電泳介紹	5
1.4.2毛細管電泳分離原理	6
1.4.3毛細管電泳分離方法	6
1.5 MALDI-TOF/MS簡介	7
1.6 測量幾丁聚醣與幾丁寡醣之去乙醯化程度	8
1.6.1以毛細管電泳測量幾丁聚醣之去乙醯化程度	8
1.6.2以MALDI-TOF/MS測量幾丁寡醣之平均去乙醯化程度	10
1.7本章參考文獻	11
第二章 實驗	14
2.1實驗藥品與器材	14
2.2實驗儀器	18
2.2.1 毛細管電泳儀	18
2.2.2基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF/MS)	18
2.2.3 酸鹼分析儀(pH meter)：	18
2.2.4冷凍乾燥機：	19
2.2.5減壓濃縮機 :	19
2.2.6離心機：	19
2.2.7振盪恆溫培養箱：	19
2.2.8去離子水處理器：	19
2.3步驟與方法	20
2.3.1毛細管處理方式	20
2.3.2電泳緩衝溶液之配置	20
2.3.3以毛細管電泳測量幾丁聚醣去乙醯化程度條件和步驟	21
2.3.4以毛細管電泳測量幾丁聚醣分子量之條件和步驟	23
2.3.5利用核磁共振法測量幾丁聚醣之去乙醯化程度	26
2.3.6利用MALDI-TOF/MS分析幾丁寡醣樣品	28
2.4實驗樣品備製	32
2.4.1幾丁聚醣之乙醯化反應	32
2.4.2幾丁聚醣與幾丁質之去乙醯化反應	33
2.4.3幾丁聚醣之降解反應	36
2.4.4幾丁聚醣降解產物之分離萃取方法	37
2.5本章參考文獻	40
第三章 結果與討論	41
3.1幾丁聚醣去乙醯化程度及其分佈之改質	41
3.1.1樣品經直接乙醯化反應	41
3.1.2樣品經去乙醯化和再乙醯化反應	47
3.2幾丁質之去乙醯化反應	49
3.2.1 幾丁質去乙醯化反應之動態研究	49
3.3幾丁質去乙醯化反應分佈	55
3.4以pepsin降解不同分子量樣品幾丁聚醣	59
3.4.1以pepsin降解幾丁聚醣所得低分子量幾丁聚醣產物之分析	59
3.4.2以pepsin降解幾丁聚醣所得幾丁寡醣產物之分析	83
3.4.3 LMwtC與chitooligomers產物DDA之比較	85
3.5幾丁聚醣經pepsin降解產物的分離萃取方法之比較	93
第四章 結論	112

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