跳到主要內容

臺灣博碩士論文加值系統

(23.20.20.52) 您好!臺灣時間:2022/01/24 19:44
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:周茂村
研究生(外文):Mao-Tsun Chou
論文名稱:不同化學變性劑、溫度和還原劑對溶菌酶結構改變之研究
論文名稱(外文):The studies of the lysozyme structure change with different chemical denaturants, temperatures and reduced reagent
指導教授:陳文逸陳文逸引用關係
指導教授(外文):Wen-yih Chen
學位類別:碩士
校院名稱:國立中央大學
系所名稱:化學工程與材料工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:125
中文關鍵詞:還原劑尿素胍鹽酸二硫代蘇糖醇圓二色光譜儀螢光光譜儀溶菌□結構變性劑溫度
外文關鍵詞:circular dichroismfluorescence spectrophotometerlysozymestructuredenaturanttemperaturereduced reagentureaguanidine hydrochloridedithiothreitol
相關次數:
  • 被引用被引用:4
  • 點閱點閱:827
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
重組蛋白質在回收過程中,常會因為胜□濃度過高而形成結構錯誤、不具活性且不溶解的聚集體。在工業上,我們常利用尿素或胍酸鹽這兩種變性劑來溶解這些聚集體,進行復性或純化分離的程序後,再以其他方式移去變性劑,以獲得所需要的蛋白質。
一般而言,穩定蛋白質結構的力量包括疏水作用力、氫鍵、靜電作用力和共價雙硫鍵。常見的變性劑中,尿素能夠破壞蛋白質的疏水作用力,胍酸鹽的攻擊對象則是疏水作用力及靜電作用力,我們有時也會添加鹽類(如氯化鈉、氯化鋰)於尿素中,以提供破壞靜電作用力的力量;還原劑二硫代蘇糖醇(DTT)則用於還原蛋白質內部的雙硫鍵。由於變性劑的作用機制特性不同,對於蛋白質結構所產生的影響也不相同。
本研究藉由添加不同濃度的尿素、胍酸鹽、鹽類、操控實驗溫度、是否添加還原劑,以圓二色光譜儀和螢光光譜儀,觀察溶菌□在不同環境下,其二級結構與三級結構的變化,探討影響蛋白質穩定之作用力與光譜結構的相關性。
在添加還原劑的情況下,對於相同濃度的不同變性劑而言,胍酸鹽無論濃度高低,其改變蛋白質結構的能力,皆比尿素及氯化鈉來得大。若未添加還原劑,則僅有高濃度的胍酸鹽可令溶菌□的結構產生顯著變化。
螢光實驗顯示,若雙硫鍵仍然存在,色胺酸不會輕易地從蛋白質內部暴露出來。此外,由溫度變化實驗,可知添加變性劑和還原劑的變性反應是一個放熱反應。系統亂度會下降,而且變性的特徵溫度也遠較在緩衝溶液環境下的變性溫度還低。
由以上實驗結果可知,若溶菌□內部的雙硫鍵未被還原,我們需使用較高濃度且能破壞疏水作用力及靜電作用力的變性劑,才可使溶菌□的結構產生顯著變化。若雙硫鍵被還原,則溶菌□結構才容易受變性劑的影響而產生變化。這顯示,溶菌□本身是一個結構相當堅硬的蛋白質。
除此之外,若輔以紫外光光譜儀測量溶菌□的活性,可瞭解酵素活性與結構之間的關係。另以恆溫滴定微卡計測量各變性環境下的反應焓值,結合光譜實驗計算所得的熱力學資料,能用以探討溶菌□變性反應的路徑是two state或multi-state 的相轉變過程。
In the process of recombinant protein production, we often get inactive and insoluble inclusion bodies instead of target proteins due to high peptide concentration. In industrial application, denaturants such as urea or guanidine hydrochloride are used to solve these inclusion bodies to renature and purify/separate proteins and then denaturants are removed to get the protein we want.
Factors to stabilize protein structures include hydrophobic interactions, hydrogen bonding, electrostatic interaction and covalent disulfide bonding. Urea is mainly used to break hydrophobic interactions and guanidine hydrochloride is to destroy hydrophobic and electrostatic interactions. Sometimes, salts such as sodium chloride/lithium chloride are added in urea as electrostatic interaction destroyer to enhance the denaturant power. Another commonly used reduced reagent (dithiothreitol, DTT) is to reduce disulfide bonding within protein. All these different mechanisms bring various influences to protein structures.
In our study, we examine secondary and tertiary structure changes of lysozyme in various conditions (different concentrations of urea / guanidine hydrochloride / salts (sodium chloride), experimental temperature, with/without reduced reagent) with the aid of circular dichroism and fluorescence spectrophotometer to investigate the relationship between above factors and spectrum structure.
When reduced reagent exists, guanidine hydrochloride causes greater changes in protein structure than urea and sodium chloride. Without reduced reagent, only high concentration of guanidine chloride causes significant protein structure changes. The results of fluorescence experiments indicates that tryptophan inside protein didn''t expose easily when disulfide bond remained unbroken.
Besides, from temperature manipulation experiments, we see the denature reaction with denaturants and reducing agents added is an exothermic reaction. At the mean time, the entropy of system decreases and melting temperature (Tm) is lower than that in buffer solution.
From the above experiments, we know that if the disulfide bond is not reduced, we have to use denaturant with the capacity to break hydrophobic and electrostatic interactions in high concentration to induce significant changes in lysozyme structure. When disulfide bond is reduced, denaturants causes changes of lysozyme structure easier that indicate lysozyme is a hard protein itself.
It’s possible to investigate the relationship between activity and structure of enzymes with the aid of ultraviolet spectrometer. In further studies, comparing reaction enthalpy in various conditions measured by isothermal titration calorimeter with thermodynamic data from spectrometer, we can learn the pathway of lysozyme denature process to see it’s two state or multi-state phase transformation reaction.
目 錄
中文摘要......................................................................Ⅰ
Abstract......................................................................Ⅲ
目錄..........................................................................Ⅴ
圖目錄........................................................................Ⅷ
表目錄......................................................................ⅩⅠ
第一章 緒論..................................................................1
第二章 實驗原理與文獻回顧....................................................3
2.1 蛋白質介紹............................................................3
2.1.1 蛋白質的組成、功能及穩定結構之作用力..............................3
2.1.2 蛋白質結構........................................................9
2.1.3 穩定態的構形與特性...............................................14
2.1.4 蛋白質摺疊模型...................................................15
2.2 蛋白質之變性.........................................................17
2.2.1 破壞蛋白質穩定力量的方式.........................................17
2.2.2 變性劑與還原劑對蛋白質分子之作用.................................18
2.3 常見的分析方法.......................................................20
2.4 圓二色光譜儀.........................................................21
2.4.1 圓二色光譜儀發展歷史.............................................21
2.4.2 光的吸收與光學活性的關係.........................................24
2.4.3 圓二色光譜儀測量原理.............................................28
2.4.4 蛋白質或多胜□的圓二色光譜.......................................33
2.5 螢光光譜儀...........................................................36
2.6 溶菌□介紹...........................................................40
2.6.1 溶菌□的發現及簡介...............................................40
2.6.2 溶菌□的結構及性質...............................................42
2.6.3 外在環境對溶菌□的影響...........................................48
2.6.4 溶菌□於圓二色光譜儀與螢光光譜儀上的研究.........................55
第三章 實驗藥品與步驟........................................................57
3.1 實驗藥品.............................................................57
3.2 實驗儀器設備.........................................................58
3.3 實驗步驟.............................................................59
3.3.1 溶液配製.........................................................59
3.3.1.1 緩衝溶液.....................................................59
3.3.1.2 變性劑溶液、還原劑溶液及蛋白質溶液...........................59
3.3.2 實驗測量.........................................................60
3.3.2.1 圓二色光譜儀實驗.............................................60
3.3.2.2 螢光光譜儀實驗...............................................61
第四章 結果與討論............................................................62
4.1圓二色光譜儀實驗......................................................62
4.2 螢光光譜儀實驗.......................................................84
4.3 溫度變化實驗.........................................................96
第五章 結論與展望...........................................................104
5.1 結論................................................................104
5.2 展望................................................................106
第六章 參考文獻.............................................................107
圖 目 錄
圖2-1 穩定蛋白質結構之作用力..................................................8
圖2-2 蛋白質各級結構.........................................................11
圖2-3 蛋白質摺疊co-operativity特性示意圖.....................................16
圖2-4 Framework model and Collapse Model.....................................16
圖2-5 Urea及GuHCl破壞蛋白質分子結構之機制....................................19
圖2-6 左右旋偏極光之觀察示意圖...............................................23
圖2-7 紫外光-可見光吸收光譜;分子電子被紫外光或可見光激發後的能階躍遷示意圖.26
圖2-8 一般有機化合物及光學活性物的吸收峰.....................................27
圖2-9 構成光的振動電場及磁場示意圖...........................................29
圖2-10 光通過平面極化器示意圖................................................29
圖2-11 光分解成左右旋圓偏極光示意圖..........................................29
圖2-12 線性偏極光通過光學活性物示意圖........................................32
圖2-13 左右旋圓偏極光的合成..................................................32
圖2-14 蛋白質在圓二色光譜儀上的二級結構吸收圖................................35
圖2-15 電子躍遷能階圖........................................................37
圖2-16 三種芳香胺基酸的吸收光譜圖;三種芳香胺基酸的螢光光譜圖................38
圖2-17 溶菌□一級結構圖......................................................43
圖2-18 溶菌□三級結構圖......................................................44
圖2-19 溶菌□水解細胞壁之b(1→4)醣□鍵結位置.................................47
圖2-20 溶菌□活性位置圖......................................................47
圖2-21 pH值對在蛋白或緩衝液中的溶菌□影響....................................50
圖2-22 在不同濃度緩衝液中溶菌□活性之變化....................................51
圖4-1 不同變性時間Near-UV全波長CD光譜圖......................................63
圖4-2 不同變性時間Far-UV全波長CD光譜圖.......................................64
圖4-3 GuHCl+32mM DTT於289nm之Near-UV CD變化量隨時間變化圖....................69
圖4-4 GuHCl+32mM DTT於222nm之Far-UV CD變化量隨時間變化圖.....................70
圖4-5 GuHCl於289nm之Near-UV CD變化量隨時間變化圖.............................71
圖4-6 GuHCl於222nm之Far-UV CD變化量隨時間變化圖..............................72
圖4-7 Urea+32mM DTT於289nm之Near-UV CD變化量隨時間變化圖.....................73
圖4-8 Urea+32mM DTT於222nm之Far-UV CD變化量隨時間變化圖......................74
圖4-9 Urea於289nm之Near-UV CD變化量隨時間變化圖..............................75
圖4-10 Urea於222nm之Far-UV CD變化量隨時間變化圖..............................76
圖4-11 NaCl+32mM DTT於289nm之Near-UV CD變化量隨時間變化圖....................77
圖4-12 NaCl+32mM DTT於222nm之Far-UV CD變化量隨時間變化圖.....................78
圖4-13 NaCl+Urea+32mM DTT於289nm之Near-UV CD變化量等高線圖...................79
圖4-14 NaCl+Urea+32mM DTT於222nm之Far-UV CD變化量等高線圖....................80
圖4-15 不同變性時間之全波長範圍螢光強度光譜圖................................85
圖4-16 GuHCl+32mM DTT於350nm螢光強度變化量隨時間變化圖.......................88
圖4-17 GuHCl於350nm螢光強度變化量隨時間變化圖................................89
圖4-18 Urea+32mM DTT於350nm螢光強度變化量隨時間變化圖........................90
圖4-19 Urea於350nm螢光強度變化量隨時間變化圖.................................91
圖4-20 NaCl+32mM DTT於350nm螢光強度變化量隨時間變化圖........................92
圖4-21 不同變性劑之CD-溫度圖................................................99
表 目 錄
表2-1 20種基本胺基酸..........................................................5
表2-2 常見的蛋白質變性方法...................................................17
表2-3 常見的分析方法.........................................................20
表2-4 研究歷史紀錄...........................................................21
表2-5 圓二色光譜上常見的發色團與最大吸收範圍.................................27
表2-6 圓二色光譜圖波峰代表意義...............................................35
表2-7 不同來源溶菌□之胺基酸組成.............................................41
表2-8 溶菌□在NaCl溶液中的穩定性.............................................53
表2-9 多醣類對溶菌□活性之影響...............................................53
表2-10 溶菌□在蔗糖溶液中的熱穩定性..........................................54
表4-1 不同變性劑環境的K、△G、△HvH、△S和Tm列表............................100
表4-2 不同變性環境下溶菌□變性反應之熱力學數據..............................103
1. Armstrong, F. B. Biochemistry. Oxford, 1989.2. Bailey, P. D. An introduction to peptide chemistry. John Wiley & Sons, Ltd., 1990.3. Berges, H., Kassah, E., Conte, D., Adjadj, E., Houee-Levin, C. "Ab-Initio calculations on arginine-disulfide complexes modeling the one-electron reduction of lysozyme. Comparison to an experimental reinvestigation." J. Phys. Chem., 101(42), 7809-7817, 1997.4. Beychok, S., Warner, R.C. "Denaturation and electrophoretic behavior of lysozyme." J. Am. Chem., 81, 1892, 1959.5. Bhattacharjya, S., Balaram, P. "Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg-white lysozyme." Protein Sci., 6, 1065-1073, 1997.6. Blake, C. C. F., Koenig, D. F., Mair, G. A., North, A. C. T., Phillips, D. C., Sarma, V. R. "Structure of hen egg-white lysozyme." Nature, 22, 757-761, 1965.7. Bovey, F.A. Polymer conformation and configuration. Academic Press Inc., 1969.8. Boyer, R. F. Biochemistry. Brooks/Cole Publishing Company, 1999.9. Campbell, M. K. Biochemistry. Saunders College Publishing, 1995.10. Canfield, R. E. "Chapter 1: Introduction", In Lysozyme. Osserman, E. F., Canfield, R. E., Beychok, S. (Eds). Academic press. 1974.11. Carrect, R. H., Grisham, C. M. Biochemistry. Saunders College Publishing. 1999.12. Copeland, R. A. Methods for protein analysis. Chapman & Hall, 1994.13. Charney, E. The molecular basis of optical activity: Optical rotatory dispersion and circular dichroism. John Wiley & Sons, Inc., 1979.14. Cunningham, F. E., Lineweaver, H. "Stabilization of egg-white proteins to pasteurizing temperatures above 60℃." Food. Technol., 19, 136, 1965.15. Crabbé, P. In ORD and CD in chenistry and biochemistry. Academic Press Inc., 1972.16. Darby, N. J., Creighton, T. E. Protein structure. IRL Press at Oxford University Press, 1993.17. Day, R. A., Underwood, A. L. Quantitative analysis. Prentice Hall, 1991.18. Dickinson, E. Matsumura, Y. "Proteins at liquid interfaces: role of molten globule state." Colloids Surfaces B., 3(1-2), 1-17, 1994.19. Dill, K.A. "Theory for the folding and stability of globular-proteins." Biochemistry, 24(6), 1501-1509, 1985.20. Fasman, G. D. Circular dichroism and the conformation analysis of biomolecules. Plenum Press. 1996.21. Fersht, A. Structure and mechanism in protein science. W. H. Freeman and Co. 508-539, 547-553, 1999.22. Freire, E. "Thermal denaturation methods in the study of protein folding." Meth. Enzymol., 259, 144-168, 1995.23. Gorin, G., Wang, S. -F., Wilson, B. M. "Assay of lysozyme by its lytic action on Micrococcus lysodeikticus cell." Anal. Biochem., 39, 113, 1971.24. Gorin, L., Felic, F. "Influence du manganede sur la stabilite du lysozyme. I. Influence du manganese ser le vitesse d'' inactivation irreversible du lysozyme par la chaleur." Biochem. Biophys. Acta., 10, 128,1953.25. Greenfield, N.J. "Review: Methods to estimate the conformation of proteins and polypeptides from circular dichroism data." Anal. Biochem., 235, 1-10, 1996.26. Hamaguchi, K. "Conformation and enzymatic activity of lysozyme." Tampakushitsu. Kakusan Kosa., 13, 98, 1968.27. Hash, J. H. "Chapter 9: Lysozyme chalarposis", In Lysozyme. Osserman, E. F., Canfield, R. E., Beychok, S. (Eds). Academic press. 1974.28. Hermans, J. "Methods for the study of reversible denaturation of proteins and interpretation of data." Methods Biochem. Anal., 13, 81-111, 1965.29. Imoto. T., Yagishita, K. "A Simple activity measurement of lysozyme." Agr. Biol. Chem., 35, 1154, 1971.30. Jirgensons, B., Philadelphia, A. K., Evanston, G. F. S., Berlin, H. G. W. Optical rotatory dispersion of proteins and other macromolecules. Springer-Verlag New York Inc., 1969.31. John, D. M., Weeks, K. M. "For the record: van''t Hoff enthalpies without baselines." Protein Sci., 9, 1416-1419, 2000.32. Karplus, M., Weaver, D. L., Nature, 260, 404-406, 1976.33. Kauzmann, W. "Some factors in the interoretation of protein denaturation." Adv. Protein Chem., 14, 1-63, 1959.34. Kuwajima, K. "The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure." Protein. Struct. Funct. Genet., 6(2), 87-103, 1989.35. Laurents, D. V., Baldwin, R. L. "Characterization of the unfolding pathway of hen egg white lysozyme." Biochemistry, 36, 1496-1504, 1997.36. Lightner, D. A., Gurst, J. E. Organic conformational analysis and stereochemistry from circular dichroism spectroscopy. John Wiley & Sons, Inc., 2000.37. Liu, Y., Sturtevant, J. M. "The observed change in heat capacity accompanying the thermal unfolding of proteins depends on the composition of the solution and on the method employed to change the temperature of unfolding." Biochemistry, 35, 3059-3062, 1996.38. Lumry, R., Bioltonen, R., Brandts, J. F. "Validity of the "two-state" hypothesis for conformational transitions of proteins." Biopolymers, 4, 917-944, 1966.39. Makki, F., Durance, T. D. "Thermal inactivation of lysozyme as influenced by pH, sucrose and sodium chloride and inactivation and preservative effect in beer." Food Res. Int., 29(7), 635-645, 1996.40. Mathews, C. K., Holde, K. E. V., Ahern, K.G. Biochemistry. Addison Wesley Longman, Inc. 2000.41. Matsubara, M., Nohara, D., Sakai, T. "Difference between guanidinium chloride and urea as denaturants of globular protein: The possibility of application to improve refolding process." Chem. Pharm. Bull., 40, 550-552, 1992.42. Mckee, T. "Biochemistry." Wm.C. Brown Publisher, 1996.43. Muesing, R. A., Nishida, T. "Disruption of low- and high-density human plasma lipoproteins and phospholipid dispersions by 1-anilinonaphthalene-8-sulfonate." Biochemistry, 10(15), 2952-2962, 1971.44. Nakanishi, K., Berova, N., Woody, R. W. Circular dichroism principles and applications. VCH Publishers, Inc., 1994.45. Neurath, H., Greenstein, J. P., Putnam, F. W., Erickson, J.O. "The chemistry of protein denaturation." Chem. Rev., 32, 157-265, 1943.46. Nölting, B. Protein folding kinetic. Springer-Verlag Berlin Heidelberg, 1999.47. Ohgushi, M., Wada, A. " ''Molten-globule state'': A compact form of globular-proteins with mobile side-chains." FEBS Lett., (164(1), 21-24, 1983.48. Osserman, E. F., Canfield, R. E., Beychok, S. "Sections 3: biological and clinical studies", in Lysozyme. Osserman, E. F., Canfield, R. E., Beychok, S. (Eds). Academic press. 1974.49. Phillips, D. C. "Chapter 2: The properties of lysozyme", in Lysozyme. Osserman, E. F., Canfield, R. E., Beychok, S. (Eds). Academic press. 1974.50. Proctor, V. A. Cunningham, F. E. "The chemistry of lysozyme and its use as a food preservative and a pharmaceutical." CRC Crit. Rev. Food Sci. Nutr., 26(4) 359-395, 1988.51. Ptitsyn, O. B. Protein folding. Creighton, T. E. (Ed.), Freeman, 243, 1992.52. Ptitsyn, O. B., Pain, R. H., Semisotnov, G. V., Zerovnik, E., Razgulyaev, O. I. "Evidence for a molten globule state as a general intermediate in protein folding." FEBS Lett., 262(1), 20-24, 1990.53. Raford, S. E., Dobson, C. M., Evans, P. A. "The folding of hen lysozyme involves partially structure intermediates and multiple pathways." Nature, 358, 302-307, 1992.54. Rodger, A., Nordén, B. "Circular dichroism and linear dichroism." Oxford, 1997.55. Rothwarf, D. M., Scheraga, H. A. "Role of non-native aromatic and hydrophobic interaction in the folding of hen egg white lysozyme." Biochemistry, 35, 13797-13807, 1996.56. Sadana, A. "Review: protein refolding and inactivation during bioseparation: Bioseparation implication." Biotechnol. Bioeng., 48(5), 481-489, 1995.57. Sandow, A. "The anti-bacterial activity of egg-white." Proc. Soc. Exp. Biol. Med., 24, 172, 1926.58. Sasahara, K., Demura, M., Nitta, K. "Partially unfolded equilibrium state of hen lysozyme studied by circular dichroism spectroscopy." Biochemistry, 39, 647506482, 2000.59. Saxena, V. P., Wetlaufer, D. B., "Formation of three-dimensional structure in proteins. I. Rapid nonenzymic reactivation of reduced lysozyme." Biochemistry, 9, 5015-5023, 1970.60. Shakhnovich, E. I., Finkelstein, A. V. "Theory of cooperative transitions in protein molecules. 1. Why denaturation of globular protein is a 1st-order phase-transition." Bioplyomers, 28(10), 1667-1680, 1989.61. Sherman, R. D., Charles, M. P. "Factors affecting the activity of egg white lysozyme." Arch. Biochem. Biophys., 40, 364, 1951.62. Smith, A. L. Shirazi, H. M., Mulligan, S. R. "Water sorption isotherms and enthalpies of water sorption by lysozyme using the quartz crystal microbalance/heat conduction calorimeter." Biochem. Biophys. Acta., 1594, 150-159, 2002.63. Summers, C. A., Flowers Ⅱ, R. A. "Protein renaturation by the liquid organic salt ethylammonium nitrate." Protein Sci., 9, 2001-2008, 2000.64. Teale, F. W., Weber, G. "Ultraviolet fluorescence of the aromatic amino acids." Biochem. J., 65, 476-482, 1957.65. Tedford, L. -A., Smith, D., Schaschke, C. T. "High pressure processing effects on the molecular structure of ovalbumin, lysozyme and b-lactoglobin." Food Res. Int., 32, 101-106, 1999.66. Townes, J. K. "Moisture content in proteins: its effects and measurement." J. Chromatogr. A., 705, 115-127, 1995.67. van den Berg, B. Chung, E. W., Robinson, C. V., Dobson, C. M. "Characterization of the dominant oxidative folding intermediate of hen lysozyme." J. Mol. Biol., 290, 781-796, 1999.68. Velluz, L., Legrand, M., Grosjean, M., Optical circular dichroism. Academic Press, Inc., 1965.69. Wescott, C. R., Klibanov, A. M. "The solvent dependence of enzyme specificity." Biochem Biophys. Acta., 1206(1), 1-9, 1994.70. West, S. M., Guise, A. D., Chaudhuri, J. B. "A comparison of the denaturants urea and guanidine hydrochloride on protein refolding." Food Bioprod. Process., 75, 50-56, 1997.71. Wetlaufer, D. B. Adv. Protein Chem., 17, 303-390, 1962.72. Yang, J. T., Wu, C. -S. C., Martinez, H.M. "Calculation of protein conformation from circular dichroism." Meth. Enzymol., 130, 208-269, 1986.73. Yashitake, S., Shinichiro, A. "Use of egg white lysozyme in the food industry." New. Food. Inc., 19, 17, 1977.74. 江建民 "應用螢光及旋光技術於生命薄膜的研究." 科儀新知, 18(6), 44-57, 1997.75. 林敬二、林宗義 儀器分析 (上冊). 美亞書版股份有限公司, 1992.76. 李雅雯 "Ribonuclease A於逆微胞系統中的復性及其界面現象之研究." 中央大學化學工程研究所碩士. 2001.77. 郭春芳 "以circular dichroism探討溫度、pH值及鹽濃度對多醣構形之影響." 台灣大學食品研究所碩士. 2000.78. 陳美方 "酸鹼度和相對黏度對於神經毒蛋白的開放與回疊路徑的探討." 台灣大學化學研究所碩士. 200080. 陳致真 "以旋光法量葡萄糖溶液濃度之統計設計與研製." 台灣大學電機工程研究所碩士. 1997.81. 孫逸民、陳玉舜、趙敏勳、謝明學、劉興鑑 儀器分析. 全盛圖書有限公司. 1997.82. 賀孝雍 儀器分析. 曉園出版社有限公司. 1991.83. 黃世權 "澱粉分解酶摺疊程序之研究." 中原大學化學工程研究所碩士. 2001.84. 程郁郁 "含假異胞嘧啶及其衍生物之去氧寡核酸鏈在水溶液中形成核酸三螺旋之研究." 中國文化大學應用化學研究所碩士. 1997.85. 董有蘭 化學與生命. 國立編譯館. 1999.86. 趙世彬、鄒煦杕、蔡東璣、陳河吉、周大中、李文齡 生物化學. 藝軒圖書出版社. 1998.87. 劉茂宏、張勝善 "雞蛋白溶菌□之分離及溶菌性之測定." 中國畜牧會誌, 23(3), 309, 1994.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
1. 鄭詩華,「農村民宿之經營及管理」,戶外遊憩研究,第五卷,第三期,第13-24頁(1990)。
2. 歐聖榮、顏宏旭,「金門地區觀光發展衝擊認知之研究」,戶外遊憩研究,第七卷,第四期,第61-89頁(1994)。
3. 黃淑清,「以方法論的觀點來看深度訪談研究法」,輔導季刊,第三十四卷,第一期,第39-45頁(1998)。
4. 黃文卿、林晏州,「深度訪談之理論與技巧--以陽明山國家公園遊園專車推動為例」,國家公園學報,第八卷,第二期,第166-178頁(1998)。
5. 陳思倫、郭柏村,「觀音山風景區居民對觀光開發影響認知之研究」,觀光研究學報,第一卷,第二期,第48-58頁(1995)。
6. 陳思倫、高麗真,「恆春鎮居民對觀光開發的社會經濟影響之認知」,世界新聞傳播學院學報,第四期,第353-376頁(1994)。
7. 陳永龍,「觀看、地方自主性與社會權力--試論觀光互動過程中的看與被看」,山海文化雙月刊,第二期,第47-51頁(1994)。
8. 范麗娟,「深度訪談簡介」,戶外遊憩研究,第七卷,第二期,第25-35頁,(1994)。
9. 侯錦雄,「觀光區的重生-永續經營的更新計劃」,戶外遊憩研究,第九卷,第四期,第51-62頁(1996)。
10. 侯錦雄,「推動休閒農業的另一種方式--民宿」,農業世界,第一百七十六期,第24-26頁(1998)。
11. 李素馨,「觀光新紀元-永續發展的選擇」,戶外遊憩研究,第九卷,第四期,第1-17頁(1996)。