利用毛細管電泳及MALDI-TOF mass研究幾丁聚醣之降解反應

Study of degradation reaction of chitosan by capillary electrophoresis and MALDI-TOF mass

張國書(Kuo-Shu Chang)

指導教授 ： 吳俊弘

淡江大學/理學院/化學學系碩士班/碩士(2013年)

https://doi.org/10.6846/TKU.2013.01162

摘要

本論文以毛細管電泳(CE)技術分別建立測量幾丁聚醣的去乙醯化程度(DDA)和分子量(Mv)的校正曲線。在測量不同DDA(26%~95%)幾丁聚醣樣品於不同濃度(25 mM~1.6 M) 毛細管電泳緩衝溶液(tris-phosphate buffer, pH = 2.00)中的電泳遷移率(μ)時，我們發現，由CE所建構的μ對DDA(NMR測量值)校正曲線在樣品之高DDA範圍(55%~95%)和低DDA範圍(26%~ 55%)有不同的線性關係，而隨著緩衝溶液濃度提高，兩線性漸趨一致，在毛細管電泳緩衝溶液達1.5 M時，兩段DDA範圍(26%~95%)即呈一線性關係。另外，毛細管電泳圖的UV吸收峰(200 nm)訊號之半高寬(W1/2, DDA)代表樣品的DDA分布情形，我們發現將樣品進行依我們所設計的去乙醯化反應，再經乙醯化反應後，可得到較小分布但DDA不變的樣品。而毛細管電泳快速和微量分析的特性，更有利於我們探討幾丁質和幾丁聚醣的去乙醯化反應之動力學。
我們也利用CE測量幾丁聚醣樣品的黏度，以Mark-Houwink equation計算其極限黏度分子量(Mv)，再測量不同Mv幾丁聚醣在毛細管凝膠電泳(CGE, 分離介質為0.3% PEO in 100 mM tris-phosphate buffer, pH = 2.00)中的電泳遷移率，建立Log(μ)對Mv的校正曲線。據此，我們探討了以過硫酸鉀(potassium persulfate, KPS)為主的化學裂解法，和以胃蛋白酶(pepsin)、脂解酶(lipase )、和胰蛋白酶(trypsin)為主的酵素消化法，對於幾丁聚醣分子量降解反應的影響。
除了以CE分析幾丁聚醣降解後的多醣產物，我們也使用MALDI-TOF MS偵測分子量降解後的幾丁寡醣產物。根據MALDI所測得的質譜圖和數據，我們可以分析降解產物的單體組成，並比較不同降解方法所得產物的異同。

關鍵字

幾丁聚醣

並列摘要

In this thesis, we used capillary electrophoresis (CE) technique to establish the respective calibration curves for the measurements of degree of deacetylation (DDA) and molecular weight (Mv) for chitosan samples. When measuring the electrophoretic mobility (μ) of chitosan samples with DDA values ranging from 26% to 95% in CE run buffers (tris-phosphate buffer, pH = 2.00) of various concentrations (25 mM~1.6M), we found that the μ-versus-DDA (NMR measured) calibration curves possess different linearity in the higher DDA region (55%~95%) and in the lower DDA region (26%~55%). Moreover, the linearity of the two regions gradually approach to each other with increasing buffer concentrations, and finally become one linear relation in the DDA range of 26%~95% when raising the CE buffer concentration up to 1.5 M. In addition, the UV absorption peak (at 200 nm) width at half maximum (W1/2, DDA) in the CE electropherogram enable us to obtain the DDA distribution in the chitosan samples. We found that chitosan with unchanged DDA but narrower DDA distribution can be generated by designed deacetylation and acetylation reactions. The high efficiency and microanalysis features of our CE based method also facilitate the kinetic studies of the deacetylation reactions for chitin and chitosan samples.
We also used CE to measure the viscosity of the chitosan samples so as to calculate the intrinsic-viscosity defined molecular weight (Mv) by the Mark-Houwink equation, and to establish the Log(μ)-versus-Mv calibration curve by measuring the electrophoretic mobility of chitosan samples with varied Mv’s in capillary gel electrophoresis (CGE, with separation medium of 0.3% PEO in 100 mM tris-phosphate buffer, pH = 2.00). By using the CGE technique we were able to study the Mv degradation reactions of chitosan by the chemical degradation method, which is mainly initiated by potassium persulfate (KPS), and by the enzymatic digestion approach, which is based on the biological enzymes such as pepsin, lipase, and trypsin.
Besides CE, we also used MALDI-TOF mass to detect the chitooligosaccharide product of the chitosan degradation reaction. According to the MALDI-TOF mass spectrums and data, we can analyze and make a comparison among the monomer compositions of the chitooligosaccharide obtained from the abovementioned different fragmentation methods.

並列關鍵字

Chitosan ； Degradation ； CE ； MALDI

參考文獻

1. Ban, E.; Choi, O. K.; Ryu, J. C.; Yoo, Y. S., “ Capillary electrophoresis

of high-molecular chitosan: the natural carbohydrate biopolymer ”,

Electrophoresis, 2001, 22, 2217-2221.

2.曾詩穎 淡江大學 化學學系碩士班 2010.

physicochemical behaviors of chitosan in aqueous Solution ”,