Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3598
標題: 疏水官能基薄膜之製備及其在純化盤尼西林醯胺酵素上之應用
Preparation of Hydrophobic Ligands Membrane and Its Application on Penicillin G Acylase Purification
作者: 陳靜雯
Chen, Chin-Wen
關鍵字: hydrophobic ligands
吡啶
pyridine
penicillin G acylase
immobilized metal affinity membrane
purification
盤尼西林醯胺酵素
固定化金屬親和薄膜
純化
出版社: 化學工程學系所
引用: [1] Knight P., Downstram Processing. Bio/technology, 1989. 7(8) 777-782. [2] Luksa J., Menart V., Milicic S., Kus B., Gaberc-Porekar V. and Josic D., Purification of Human Tumour Necrosis Factor by Membrane Chromatography. Journal of Chromatography A, 1994. 661(1-2) 161-168. [3] Ghosh R., Protein Separation Using Membrane Chromatography: Opportunities and Challenges. Journal of Chromatography A, 2002. 952(1-2) 13-27. [4] 陳東煌, 離子交換膜的應用. 化工, 1999. 46(4) 14-23 [5] Freitag R., Splitt H. and Reif O.-W., Controlled Mixed-Mode Interaction Chromatography on Membrane Adsorbers. Journal of Chromatography A, 1996. 728(1-2) 129-137. [6] Langlotz P. and Kroner K. H., Surface-Modified Membranes as a Matrix for Protein Purification. Journal of Chromatography A, 1992. 591(1-2) 107-113. [7] Queiroz J. A., Tomaz C. T. and Cabral J. M. S., Hydrophobic Interaction Chromatography of Proteins. Journal of Biotechnology, 2001. 87(2) 143-159. [8] Coen C. J., Blanch H. W. and Prausnitz J. M., Salting out of Aqueous Proteins: Phase Equilibria and Intermolecular Potentials. AIChE Journal, 1995. 41(4) 996-1004. [9] 王銀善, 疏水層析, 生物化學技術與原理, 趙永芳, Editor. 2002, 科學出版社: 北京. p. 65. [10] Pu Y. and Wang Z.-X., Advancement of Ion Exchange Chromatography and Hydrophobic Interaction Chromatography Media Application in Protein Chromatography. Chinese Journal of Biotechnology, 2004. 20(6) 975-982. [11] Diogo M. M., Silva S., Cabral J. M. S. and Queiroz J. A., Hydrophobic Interaction Chromatography of Chromobacterium Viscosum Lipase on Polypropylene Glycol Immobilised on Sepharose. Journal of Chromatography A, 1999. 849(2) 413-419. [12] Tomaz C. T., Duarte D. and Queiroz J. A., Comparative Study on the Fractionation of Cellulases on Some Hydrophobic Interaction Chromatography Adsorbents. Journal of Chromatography A, 2002. 944(1-2) 211-216. [13] Porath J., Maisano F. and Belew M., Thiophilic Adsorption - a New Method for Protein Fractionation. FEBS Letters, 1985. 185(2) 306-310. [14] Porath J. and Oscarsson S., A New Kind Of "Thiophilic" Electron-Donor-Acceptor Adsorbent. Die Makromolekulare Chemie. Macromolecular Symposia, 1988. 17 359-371. [15] Berna N., Berna P. and Oscarsson S., Cosolvent-Induced Adsorption and Desorption of Serum Proteins on an Amphiphilic Mercaptomethylene Pyridine-Derivatized Agarose Gel. Archives of Biochemistry and Biophysics, 1996. 330(1) 188-192. [16] Berna P. P., Berna N., Porath J. and Oscarsson S., Comparison of the Protein Adsorption Selectivity of Salt-Promoted Agarose-Based Adsorbents: Hydrophobic, Thiophilic and Electron Donor-Acceptor Adsorbents. Journal of Chromatography A, 1998. 800(2) 151-159. [17] Xue B., Ersson B., Porath J. and Caldwell K., Chromatographic and Fluorometric Study of Interactions between Thiophilic and Hydrophobic Ligands and Tryptophan Peptide Homologues. Journal of Chromatography A, 2006. 1107(1-2) 46-51. [18] Wang Y., Wang Q., Luo X., Guan G., Wei Z. and Yan K., Study on Purif Ication Process of Recombinant Hbsag. Chinese Journal of Biotechnology, 1999. 15 263-266. [19] Burton S. C. and Harding D. R. K., Hydrophobic Charge Induction Chromatography: Salt Independent Protein Adsorption and Facile Elution with Aqueous Buffers. Journal of Chromatography A, 1998. 814(1-2) 71-81. [20] Guerrier L., Flayeux I. and Boschetti E., A Dual-Mode Approach to the Selective Separation of Antibodies and Their Fragments. Journal of Chromatography B: Biomedical Sciences and Applications, 2001. 755(1-2) 37-46. [21] Schwartz W., Judd D., Wysocki M., Guerrier L., Birck-Wilson E. and Boschetti E., Comparison of Hydrophobic Charge Induction Chromatography with Affinity Chromatography on Protein a for Harvest and Purification of Antibodies. Journal of Chromatography A, 2001. 908(1-2) 251-263. [22] Boschetti E., Antibody Separation by Hydrophobic Charge Induction Chromatography. Trends in Biotechnology, 2002. 20(8) 333-337. [23] Perkins T. W., Mak D. S., Root T. W. and Lightfoot E. N., Protein Retention in Hydrophobic Interaction Chromatography: Modeling Variation with Buffer Ionic Strength and Column Hydrophobicity. Journal of Chromatography A, 1997. 766(1-2) 1-14. [24] Voute N., Guerrier L. and Santambien P., Specific Capture of Antibodies Using Hydrophobic Charge Induction Chromatography (Mep Hypercel). in The 20th International Symposium on the Separation and Analysis of Proteins, Peptides, and Polynucleotides. 2000: Ljubljana, SLOVENIA. [25] Guerrier L., Girot P., Schwartz W. and Boschetti E., New Method for the Selective Capture of Antibodies under Physiolgical Conditions. Bioseparation, 2000. 9(4) 211-221. [26] Weatherly G. T., Bouvier A., Lydiard D. D., Chapline J., Henderson I., Schrimsher J. L. and Shepard S. R., Initial Purification of Recombinant Botulinum Neurotoxin Fragments for Pharmaceutical Production Using Hydrophobic Charge Induction Chromatography. Journal of Chromatography A, 2002. 952(1-2) 99-110. [27] Willems A., Leoen J., Schoonooghe S., Grooten J. and Mertens N., Optimizing Expression and Purification from Cell Culture Medium of Trispecific Recombinant Antibody Derivatives. Journal of Chromatography B, 2003. 786(1-2) 161-176. [28] Sulkowski E., Purification of Proteins by Imac. Trends in Biotechnology, 1985. 3(1) 1-7. [29] Hemdan E. S. and Porath J., Development of Immobilized Metal Affinity Chromatography : Ii. Interaction of Amino Acids with Immobilized Nickel Iminodiacetate. Journal of Chromatography A, 1985. 323(2) 255-264. [30] Tanford C., The Hydrophobic Effect : Formation of Micelles and Biological Membranes. 1980, New York: Wiley-Interscience. [31] Creighton T. E. and Freeman W. E., Proteins, Structure and Molecular Properties. 1984, New York. [32] Porath J., Salt-Promoted Adsorption: Recent Developments. Journal of Chromatography: Biomedical Applications, 1986. 376 331-341. [33] Hofstee B. H. J., Hydrophobic Affinity Chromatography of Proteins. Analytical Biochemistry, 1973. 52(2) 430-448. [34] Shaltiel S. and William B. J., Hydrophobic Chromatography. Methods in Enzymology, 1984. 104 69-96. [35] Melander W. and Horvath C., Salt Effects on Hydrophobic Interactions in Precipitation and Chromatography of Proteins: An Interpretation of the Lyotropic Series. Archives of Biochemistry and Biophysics, 1977. 183(1) 200-215. [36] Arai T. and Norde W., The Behavior of Some Model Proteins at Solid-Liquid Interfaces 1. Adsorption from Single Protein Solutions. Colloids and Surfaces, 1990. 51 1-15. [37] Kato Y., Nakamura K., Kitamura T., Moriyama H., Hasegawa M. and Sasaki H., Separation of Proteins by Hydrophobic Interaction Chromatography at Low Salt Concentration. Journal of Chromatography A, 2002. 971(1-2) 143-149. [38] Besseling N. A. M., Theory of Hydration Forces between Surfaces. Langmuir, 1997. 13(7) 2113-2122. [39] Dill K. A., Dominant Forces in Protein Folding. Biochemistry, 1990. 29(31) 7133-7155. [40] Norde W. and Haynes C. A., Reversibility and the Mechanism of Protein Adsorption. In Proteins at Interface Ii. Fundamentals and Applications, Acs Symposium, T.A. Horbett and J.L. Brash, Editors. 1995, American Chemical Society: Washington, DC. p. 27-40. [41] Esquibel-King M. A., Dias-Cabral A. C., Queiroz J. A. and Pinto N. G., Study of Hydrophobic Interaction Adsorption of Bovine Serum Albumin under Overloaded Conditions Using Flow Microcalorimetry. Journal of Chromatography A, 1999. 865(1-2) 111-122. [42] Hensgens C. M. H., Keizer E., Snijder H. J. and Dijkstra B. W. Penicillin Acylase, Mutant 2001 [Available from http://www.rcsb.org/pdb/explore.do?structureId=1JX9] [43] Fitton V. and Santarelli X., Evaluation of Immobilized Metal Affinity Chromatography for Purification of Penicillin Acylase. Journal of Chromatography B: Biomedical Sciences and Applications, 2001. 754(1) 135-140. [44] Sanchez J., Verdoni N., Fitton V. and Santarelli X., Efficient Two-Step Chromatographic Purification of Penicillin Acylase from Clarified Escherichia Coli Ultrasonic Homogenate. Journal of Chromatography B: Biomedical Sciences and Applications, 2001. 753(1) 45-50. [45] Liu Y.-C., ChangChien C.-C. and Suen S.-Y., Purification of Penicillin G Acylase Using Immobilized Metal Affinity Membranes. Journal of Chromatography B, 2003. 794(1) 67-76. [46] Coulon D., Cabanne C., Fitton V., Noubhani A. M., Saint-Christophe E. and Santarelli X., Penicillin Acylase Purification with the Aid of Hydrophobic Charge Induction Chromatography. Journal of Chromatography B, 2004. 808(1) 111-115. [47] Santarelli X., Fitton V., Verdoni N. and Cassagne C., Preparation, Evaluation and Application of New Pseudo-Affinity Chromatographic Supports for Penicillin Acylase Purification. Journal of Chromatography B: Biomedical Sciences and Applications, 2000. 739(1) 63-72. [48] Oscarsson S. and Porath J., Protein Chromatography with Pyridine- and Alkylthioether-Based Agarose Adsorbents. Journal of Chromatography A, 1990. 499 235-247. [49] Oscarsson S., Medin A. and Porath J., Kinetic and Conformational Factors Involved in Chemisorption and Adsorption of Proteins on Mercaptopyridine-Derivatized Agarose. Journal of Colloid and Interface Science, 1992. 152(1) 114-124. [50] Porath J., Sundberg L., Fornstedt N. and Olsson I., Salting-out in Amphiphilic Gels as a New Approach to Hydrophobia Adsorption. Nature, 1973. 245(5426) 465-466. [51] Chen C.-I., Chen C.-W., Huang C.-W. and Liu Y.-C., Simultaneous Purification and Immobilization of Penicillin G Acylase Using Bifunctional Membrane. Journal of Membrane Science, 2007. 298(1-2) 24-29. [52] 姚呈儒, 利用金屬親和薄膜固定化盤尼西林醯胺酵素之穩定性及再生性探討. 國立中興大學碩士學位論文, 2007. [53] Bradford M. M., A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 1976. 72 248. [54] Hu H.-L., Wang M.-Y., Chung C.-H. and Suen S.-Y., Purification of Vp3 Protein of Infectious Bursal Disease Virus Using Nickel Ion-Immobilized Regenerated Cellulose-Based Membranes. Journal of Chromatography B, 2006. 840(2) 76-84. [55] Pavia D. L., Lampman G. M. and Kriz G. S., Introduction to Spectroscopy : A Guide for Students of Organic Chemistry. Third Edition ed. 2001, USA. Appendix 1. [56] van Oss C. J., Good R. J. and Chaudhury M. K., Nature of the Antigen-Antibody Interaction: Primary and Secondary Bonds: Optimal Conditions for Association and Dissociation. Journal of Chromatography: Biomedical Applications, 1986. 376 111-119. [57] Melander W. R., El Rassi Z. and Horvath C., Interplay of Hydrophobic and Electrostatic Interactions in Biopolymer Chromatography : Effect of Salts on the Retention of Proteins. Journal of Chromatography A, 1989. 469 3-27. [58] Product Note: Mep Hypercel
摘要: 本研究應用再生纖維薄膜(Regenerated cellulose membrane,RCM)為固體基材,使用前以epichlorohydrin (EPI)修飾RCM,再以化學合成方式接上吡啶衍生物(Pyridine derivatives, PDs),即為疏水官能基薄膜(Hydrophobic ligands membrane,HLM),並利用HL膜來純化盤尼西林醯胺酵素(Penicillin G acylase,PGA)。 首先挑選三種含疏水官能基之PDs:2-mercaptopyridine (2-MP)、3-picolylchloride (3-PC)及4-(mercaptoethylmpyridine (4-MEP)來製備HL膜,由實驗發現4-MEP對PGA有較佳之特異性吸附(純化倍率為1.91)。因此進一步探討在膜上反應4-MEP之最佳條件,其結果為:2.54 g 4-MEP添加於25 ml, 1 M Na2CO3中,反應條件為60°C, 24 h, 150 rpm。接下來的研究發現修飾有1,8-diaminoctane延伸臂可增加4-MEP對PGA親和力,得到最佳之PGA純化倍率為2.80。使用SEM分析薄膜表面型態,改質前後的薄膜有相似的結構和孔洞分佈。 以此最適化條件製作之HLM探討PGA純化之最佳條件,其結果為: 25 ml, 50 mM Phosphate buffer (pH 6)溶液中含PGA(活性0.2 IU/ml)和2 M (NH4)2SO4,與一片HL膜吸附12小時,接著再使用10 ml, 50 mM Acetate buffer (pH 4),內含1 M NH4Cl,將膜上之PGA脫附,此操作之PGA純化倍率為3倍。 使用本實驗室研發之固定化金屬親和薄膜,將PGA粗酵素液進行第一步純化,再使用HLM進行第二階段純化PGA酵素,經由二階段純化步驟,PGA純化倍率可達50.62倍,回收率為45.4%。使用SDS-PAGE分析蛋白質,可看出許多雜蛋白已被分離,證明二階段式純化PGA之可行性。
The regenerated cellulose (RC) membrane as solid matrix was modified by epichlorohydrin (EPI), followed by constructing the pyridine derivatives (PDs) as the hydrophobic ligand. The modified membrane named as hydrophobic ligands membrane (HLM) was used to apply on penicillin G acylase (PGA) purification. Three kinds of PDs: 2-mercaptopyridine (2-MP), 3-picolylchloride (3-PC) and 4-mercaptoethylpyridine (4-MEP) was selected as hydrophobic ligands to construct the HLM. The HLM with 4-MEP ligand for PGA purification having the highest purification fold (1.91 times) was selected in this study.The optimal preparation conditions were concluded as follows: for one RC-EPI membrane, 2.54 g 4-MEP and 25 ml 1 M Na2CO3 were added and the reaction conditions were carried out at 60C, 150 rpm for 24h. Further study showed that adding a spacer arm in the modification step could incrase the HLC affinity to PGA. From the surface morphologies scanned by SEM, the original membrane and the modified membrane had the similar structure and pore distribution. In order to evaluate the HLM for purification of PGA, the crude enzymes were adsorbed to the HLM in the presence of 25ml, 50 mM phosphate buffer (pH 6) containing 2 M (NH4)2SO4, After that, 10 ml, 1 M NH4Cl and 50 mM acetate buffer (pH 4) were used to elute PGA and a 3.03-fold PGA purification was obtained. A two-stage purification was presented by combining the immobilized metal affinity membrane and HLM for PGA purification. Under the elution conditious, a 50.62-fold PGA purification and 45.4% recovery were obtained.
URI: http://hdl.handle.net/11455/3598
其他識別: U0005-0208200719043400
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0208200719043400
Appears in Collections:化學工程學系所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.