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系統識別號 U0002-1208201310020300
中文論文名稱 以毛細管電泳與基質輔助雷射脫附游離飛行時間質譜儀分析幾丁聚醣之酵素降解產物
英文論文名稱 Analysis of enzymatic degradation of chitosan by capillary electrophoresis and MALDI-TOF/MS
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 101
學期 2
出版年 102
研究生中文姓名 高振堯
研究生英文姓名 Chen-Yao Kao
電子信箱 lovepp770529@hotmail.com
學號 600160419
學位類別 碩士
語文別 中文
口試日期 2013-07-18
論文頁數 139頁
口試委員 指導教授-吳俊弘
委員-陳灝平
委員-鄧金培
委員-吳俊弘
中文關鍵字 幾丁聚醣  幾丁寡醣  降解  毛細管電泳  基質輔助雷射脫附游離飛行時間質譜儀 
英文關鍵字 chitosan  chitooligosaccharides  degradation  capillary electrophoresis  MALDI-TOF/MS 
學科別分類 學科別自然科學化學
中文摘要 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。
英文摘要 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.
論文目次 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
參考文獻 第一章
1. Tsaih, M. L.; Chen, R. H., “ The effect of reaction time and temperature during heterogenous alkali deacetylation on degree of deacetylation and molecular weight of resulting chitosan ”,
Journal of Applied Polymer Science, 2003, 88, 2917-2923.
2. Zhen, L.; Tao, G.; Xun , S.; James, Z.T.; Zhirong, Z. “Chitosan oligomers as drug carriers for renal delivery of zidovudine”, Carbohydrate polymers, 2012, 87, 2284-2290.
3. Park, J. K.;Chung, M. J.;Choi, H. N.; Park Y. I., “Effects of the Molecular Weight and the Degree of Deacetylation of Chitosan Oligosaccharides on Antitumor Activity”, Interational Journal of Molecular Sciences, 2011, 12, 266-277.
4. Stephane, T. ; Catherine, L.; Frederic, D.; Alain, D.“Chemical Preparation and Structural Characterization of a Homogeneous Series of Chitin/Chitosan Oligomers”, Biomacromolecular, 2008, 9, 1731-1738.
5. 張國書 淡江大學 化學系碩士班論文 2013.
6. 曾詩穎 淡江大學 化學系碩士班論文 2010.
7. Ravindra, R.; Krovvidi, K. R.; Khan, A. A., “ Solubility parameter of chitin and chitosan”, Carbohydrate Polymers, 1998, 36, 121-127.
8. 朱怡靜 國立聯合大學 化學工程學系碩士班 2006.
9. Yi, H.;Wu, L. Q.; Bentley, W. E.; Ghodssi, R.; Rubloff, G.W.; Culver, N.,“Biofabrication with chitosan”, Biomacromolecules, 2005, 6, 2881-2894.

10. Somorin, O.; Nishi, N.; Tokura, S.; Noguchi, J., “ Studies on
chitin. II. Preparation of benzyl and benzoylchitins ”, Polymer
Journal, 1979, 11, 391-396.
11. David, L. N.; Michael, M. C., “Lehninger Principles of biochemistry fourth edition”, Freeman.
12. Robert, S.; Gregor, M.; Tillmann, R.; Heinz, J. T. “Processing of Pestivirus Polyprotein: Cleavage Site between Autoprotease and Nucleocapsid Protein of Classical Swine Fever Virus”, Journal of Virology, 1993, 67, 7088-7095.
13. Godfrey, T. ; Reichelt, J.“Industrial Enzymology”, Macmillan Publishers Ltd, 1983
14. Carlos, A. C.; Krzysztof, N. W.; Jorge, W. C., “Pineapple fruit bromelain affinity to different protein substrates”, Food Chemistry, 2012, 133, 631-635.
15. Sono, T. ; Yokoyama, M., “Inhibitory effect of proteolytic enzymes on platelet aggregation induce by ADP or thrombin”, Experientia(Basel), 1971, 22, 1179-1181.
16. Lee, L. F.;Maskat, W. K. ;Rosli, Md. I. ; Suraini, A. A.; Kamarulazaman, K.; Osman, H., “Partial Depolymerization of Chitosan with Aid of Bromelain”, Pakistan Journal of Biological Sciences, 2005, 8(1), 73-77.
17. Castellanos, O. F.;Sinitsyn, A. P.; Vlasenko, E. Yu., “Comparative evaluation of hydrolytic efficiency toward microcrystalline cellulose of Penicillium and Trichoderma cellulases”, Bioresource Technology , 1995, 52, 119-124.
18. Bhat, M. K., “Cellulases and related enzymes in biotechnology”, Biotechnology Advances, 2000, 18, 355-383.
19. Yu, X.; Hu, J.; Ya, W.; Hong, X., “Preparation of chitooligosaccharides by the enzymatic hydrolysis of chitosan” Polymer Degradation and Stability, 2009, 94, 1895-1899.
20. Neena, N. G., “Applications of Lipase”, Journal of the American Oil Chemists' Society, 1997, 74, 621-634.
21. Houde, A.; Kademi, A.; Leblanc, D., “Lipases and Their Industrial Applications”, Applied Biochemistry and Biotechnology, 2004. 118, 155-170.
22. Roncal, T.; Oviedo, A.; Lopez de Armentia, Iratxe; Fernandez, Laura; Villaran, M. Carmen. “High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan”, Carbohydrate Reserch, 2007, 342, 2750-2756.
23. Gertner, C.; Pelaez, C. A.; Lopez B. L., “Characterization of chitin and chitosan extracted from shrimp shells by two methods”, e-Polymers, 2010, 069, 1-16.
24. John, W. F.; William L. S.; Glenn G. W., “Removal of Chill Haze from Beer with Papain Immobilized on Chitin”, Biotechnology and Bioengineering, 1977, 19, 1895-1897.
25. Vishu Kumar, A. B.;Tharanathan, R. N., “A comparative study on depolymerization of chitosan by proteolytic enzymes”, Carbohydrate Polymers, 2004, 58, 275-283.

26. Zeng, H. Q.; Wang, S. M.; Huang, J. F.; Zhuo, L. H.; Guo, Y. C., “Study on the heterogeneous degradation of chitosan with hydrogen peroxide under the catalysis of phosphotungstic acid”, Carbohydrate Polymers, 2007, 68, 761–765.
27. Juan, C. C., Pierre V. C., “Preparation of chitooligosaccharides with degree of polymerization higher than 6 by acid or enzymatic degradation of chitosan”, Biochemical Engineering Journal, 2005, 25, 165-172.
28. Hsua, S. C.; Don T.M.; Chiua, W. Y., “Free radical degradation of chitosan with potassium persulfate”, Polymer Degradation and Stability, 2002, 75, 73-83.
29. Jaroslaw M. W., Stephen G. Y., “Green molecular weight degradation of chitosan using microwave irradiation”, Polymer Degradation and Stability, 2013, 98,863-867.
30. Sun, C. M.; Qu, R. J.;Chen, H.; Ji, C. N.; Wang, C. H.; Sun, Y. Z.; Wang, B. H. “Degradation behavior of chitosan chains in the green synthesis of gold nanoparticles”, Carbohydrate Research, 2008, 343, 2595-2599.
31. Zhang, H.; Du, Y.; Yu, X. J.; Masaru, M.; Aiba, S., “Preparation of chitooligosaccharides from chitosan by a complex enzyme”, Carbohydrate Research, 1999, 320, 257-260.
32. Lemos, N. P.; Bortolotti, F.; Manetto, G.; Anderson, R. A. ; Cittadini, F.; Tagliaro, F., “Capillary electrophoresis: a new tool in forensic medicine and science ”, Science & Justice, 2001, 41, 203-210.
33. Tagliaro, F.; Turrina, S.; Smith, F. P., “Capillary electrophoresis: principles and applications in illicit drug analysis ”, Forensic Science Internationa, 1996, 77, 211-229.
34. Suntornsuk, L., “Capillary electrophoresis of phytochemical substances ”, Journal of Pharmaceutical and Biomedical Analysis, 2002, 27, 679-698.
35. Lindeberg, J., “ Capillary electrophoresis in food analysis ”, Food Chemistry, 1996, 55, 73-94.
36. Anastos, N.; Barnett, N. W.; Lewis, S. W., “ Capillary electrophoresis for forensic drug analysis: A review ”, Talanta, 2005, 67, 269-279.
37. Karas, M.; Hillenkamp, F., “Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons”, Anal. Chem., 1988, 60, 2299-2301.
38. Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yosida, Y.; Yoshida, T., “Protein and polymer analysis up to m/z 100000 by laser ionization time-of-flight mass spectrometry”, Rapid Commun. Mass Spectrom, 1988, 2, 151-158.
39. Claydon, M. A.; Davey, S. N.; EdwardsJones, V.; Gordon, D. B., “The rapid identification of intact microorganisms using mass spectrometry”, Nat. Biotechnol, 1996, 14, 1584-1586.
40. Holland, R. D.; Wilkes, J. G.; Rafii, F.; Sutherland, J. B.; Persons, C. C.; Voorhees, K. J.; Lay, J. O., “Rapid identification of intact whole bacteria based on spectral patterns using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry”, Rapid Commun. Mass Spectrom, 1996, 10, 1227-1232.
41. Krishnamurthy, T.; Ross, P. L.; Rajamani, U., “Detection of pathogenic and non-pathogenic bacteria by matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry”, Rapid Commun. Mass Spectrom, 1996, 10, 883-888.
42. 圖取自於布魯克(Brucker)公司2012年MALDI質譜儀教育訓練。
43. 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.
44. Fu, X. F.; Huang, L.; Zhai, M. L.; Li, W.; Liu, H. W., “Analysis of natural carbohydrate biopolymer-high molecular chitosan and carboxymethyl chitosan by capillary zone electrophoresis”, Carbohydrate Polymers, 2007, 68, 511-516.
45. Hattori, T.; Anraku, N.; Kato, R., “Capillary electrophoresis of chitooligosaccharides in acidic solution: Simple determination using a quaternary-ammonium-modified column and indirect photometric detection with Crystal Violet”, Journal of Chromatography B, 2010, 878, 477-480.
46. Beaudoin, M. E.; Gauthier, J.; Boucher, I.; Waldron, K. C., “Capillary electrophoresis separation of a mixture of chitin and chitosan oligosaccharides derivatized using a modified fluorophore conjugation procedure”, Journal of Separation Science, 2005, 28, 1390-1398.

47. Kuo, C.Y.; Wang, S. H.; Lin C. C.; Liao, S. K. S.; Hung, W. T.; Fang J. M.; Yang W. B., “Application of 2,3-Naphthalenediamine in Labeling Natural Carbohydrates for Capillary Electrophoresis”, Molecules, 2012, 17, 7387-7400.
48. Lavertu, M.; Xia Z.; Serreqi, A.N.; Berrada, M.; Rodrigues, A.; Wang,
D.; Buschmann, M.D.; Gupta, A., “ A validated 1H NMR method for
the determination of the degree of deacetylation of chitosan ”,Journal of Pharmaceutical and Biomedical Analysis, 2003, 32, 1149-1158.
49. Wang, W.; Bo, S.; Li, S.; Qin, W., “ Determination of the
Mark-Houwink equation for chitosans with different degrees of
deacetylation ”, International Journal of Biological Macromolecules, 1991, 13, 281-285.
50. 黃詩婷 專題研究報告 2009
第二章
1. 曾詩穎 淡江大學 化學系碩士班 2010.
2. 張國書 淡江大學 化學系碩士班 2013.
3. Wang, W.; Bo, S.; Li, S.; Qin, W., “ Determination of the
Mark-Houwink equation for chitosans with different degrees of
deacetylation ”, International Journal of Biological Macromolecules, 1991, 13, 281-285.
4. Lavertu, M.; Xia Z.; Serreqi, A.N.; Berrada, M.; Rodrigues A.; Wang,
D.; Buschmann, M.D.; Gupta, A., “ A validated 1H NMR method for
the determination of the degree of deacetylation of chitosan ”,Journal of Pharmaceutical and Biomedical Analysis, 2003, 32, 1149-1158.
5. Qun, G.; Ajun, W.; Yong, Z., “ Effect of reacetylation and degradation on the chemical and crystal structures of chitosan”, Journal of Applied Polymer Science, 2007, 104, 2720-2728.
6. Hsu, S.C.; Don, T.M.; Chiu, W.Y., “ Free radical degradation of chitosan with potassium persulfate”, Polymer Degradation and
Stability, 2002, 75, 73-83.
7. Roncal, Tomas; Oviedo, Alberto; Lopez de Armentia, Iratxe; Fernandez, Laura; Villaran, M. Carmen. “High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan”, Carbohydrate Reserch, 2007, 342, 2750-2756.
8. 周敬軒 淡江大學 化學系碩士班 2013.
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.
2. Yi, H; Wu, L. Q.; Bentley, W. E.; Ghodssi, R.; Rubloff, G.W.; Culver, N.,“Biofabrication with chitosan”, Biomacromolecules, 2005, 6, 2881-2894.
3. Susan, M., “ Study Guide with Solutions Manual for McMurry's Organic Chemistry, 7th”, Brooks Cole, 2007.
4. Park ,J. K.; Chung, M. J.; Choi, H. N.; Park Y. I., “Effects of the Molecular Weight and the Degree of Deacetylation of Chitosan Oligosaccharides on Antitumor Activity”, International Journal of Molecular Sciences, 2011, 12, 266-277.
5. 張國書 淡江大學 化學系碩士班論文 2013.
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