Title

疏水官能基玻璃纖維薄膜之製備及其固定盤尼西林醯胺酵素之穩定性

Translated Titles

Preparation of Hydrophobic Ligands on Glass Fiber Membrane and Its Stability of Penicillin G Acylase immobilization

DOI

10.6845/NCHU.2009.01012

Authors

陳善群

Key Words

疏水官能基 ; 玻璃纖維薄膜 ; 4-硫醇基-乙基吡啶 ; 盤尼西林醯胺酵素 ; 疏水作用層析 ; hydrophobic ligand ; glass fiber membrane ; penicillin G acylase ; 4-mercaptoethylpyridine ; hydrophobic interaction chromatography

PublicationName

中興大學化學工程學系所學位論文

Volume or Term/Year and Month of Publication

2009年

Academic Degree Category

碩士

Advisor

劉永銓

Content Language

繁體中文

Chinese Abstract

本研究主要在探討採用玻璃纖維薄膜(Glass fiber membrane, GFM)為固體基材,先以epichlorohydrin (EPI)修飾後,再以化學合成方式接上4-硫醇基-乙基吡啶(4-mercaptoethylmpyridine, 4-MEP),此為疏水官能基玻璃纖維薄膜(Hydrophobic ligands on glass fiber membrane, HL-GFM),並利用此HL-GFM將盤尼西林醯胺酵素(Penicillin G acylase, PGA)固定化,以探討其吸附穩定性。 為達到活化玻璃纖維薄膜目的,進行薄膜表面前處理最適條件之探討,結果以GFM浸置Piranha Solution: H2SO4/ H2O2 (30%)=7/3 (V/V)加熱95 °C、1 hr之前處理條件為最適當,上述前處理可以使GFM表面的-OH基增加。 經EPI活化處理之薄膜表面,再接上4-MEP以改質GFM之試驗,其結果為將5 ml EPI添加於25 ml , 1.4 M NaOH中,反應條件為24°C、5 hr、 150 rpm。再以4-MEP(1.6585 g)添加於25 ml, 1 M Na2CO3中,反應條件為24 °C、4 hr、150 rpm,可得到製作疏水官能基玻璃纖維薄膜的最佳條件。 使用疏水官能基玻璃纖維薄膜對PGA以疏水作用層析法(Hydrophobic interaction chromatography, HIC)吸附的最適化條件為:25 ml,50 mM phosphate buffer(pH 7.0)溶液中含PGA(活性0.1 IU/ml)及2 M (NH4)2SO4,與一片改質GFM吸附12小時,此操作之PGA固定在膜上的回復倍率最高為41.11倍。 疏水官能基玻璃纖維薄膜在吸附作用上導致PGA酵素不易脫附,吸附後薄膜上的PGA在不同的環境下,以溫度24 °C、pH 7.0相對其他酸鹼環境可維持較長的時間不易衰退,其PGA殘存活性可維持在61.5%以上。

English Abstract

The glass fiber membrane (GFM) as solid matrix was modified by epichlorohydrin (EPI), followed by constructing the 4-mercaptoethylpyridine (4-MEP) as the hydrophobic ligand. The modified membrane named as hydrophobic ligands on glass fiber membrane (HL-GFM) was applied on the immobilization of penicillin G acylase (PGA). At first, the surface of glass fibre membrane was modified, in the process of coupling EPI, the pretreatment is to immerse the membrane in Piranha solution: (H2SO4 /H2O2 (30%)=7/3(V/V)) 95 °C for 1 hr to create -OH functional groups of GFM surface. The reaction conditions of 4-MEP on GFM surface were concluded as follows:5 ml EPI in 25 ml, 1.4 M NaOH、24 °C、150 rpm for 5 hr. And then by the addition of 4-MEP (1.6585 g) in 25 ml, 1 M Na2CO3. The optimum condition of the reaction conditions were at 24°C、150 rpm for 4 hr. In order to evaluate the Hydrophobic ligands on glass fiber membrane for immobilization of PGA with hydrophobic interaction, the crude PGA enzyme was adsorbed to the modified membranes in 25 ml, 50 mM phosphate buffer (pH 7.0) containing 2 M (NH4)2SO4, After that, PGA was immobilized on membrane and a 41.1-fold PGA recovery fold was obtained. The hydrophobic ligands on glass fiber membrane leads to the adsorbed PGA enzyme difficult to elute. Immobilized PGA membrane at 24°C、pH 7.0 can be reserved for a longer time without decay as compared to that under other conditions. 61.5% PGA activity can be preserved.

Topic Category 工學院 > 化學工程學系所
工程學 > 化學工業
Reference
  1. 1. Knight, P., Downstram Processing. Bio/technology, 1989. 7(8): p. 777-782.
    連結:
  2. 2. Luksa, J., Menart, V., Milicic, S. and 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): p. 161-168.
    連結:
  3. 3. Ghosh, R., Protein separation using membrane chromatography: opportunities and challenges. Journal of Chromatography A, 2002. 952(1-2): p. 13-27.
    連結:
  4. 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): p. 129-137.
    連結:
  5. 6. Langlotz, P. and Kroner, K.H., Surface-modified membranes as a matrix for protein purification. Journal of Chromatography A, 1992. 591(1-2): p. 107-113.
    連結:
  6. 7. Queiroz, J.A., Tomaz, C.T. and Cabral, J.M.S., Hydrophobic interaction chromatography of proteins. Journal of Biotechnology, 2001. 87(2): p. 143-159.
    連結:
  7. 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): p. 996-1004.
    連結:
  8. 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): p. 413-419.
    連結:
  9. 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): p. 211-216.
    連結:
  10. 13. Porath, J., Maisano,F. and Belew, M., Thiophilic adsorption - a new method for protein fractionation. FEBS Letters, 1985. 185(2): p. 306-310.
    連結:
  11. 14. Porath, J. and Oscarsson, S., A New Kind of "Thiophilic" Electron-Donor-Acceptor Adsorbent. Die Makromolekulare Chemie. Macromolecular Symposia 1988. 17: p. 359-371.
    連結:
  12. 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): p. 188-192.
    連結:
  13. 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): p. 151-159.
    連結:
  14. 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): p. 46-51.
    連結:
  15. 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): p. 71-81.
    連結:
  16. 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): p. 37-46.
    連結:
  17. 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): p. 251-263.
    連結:
  18. 22. Boschetti, E., Antibody separation by hydrophobic charge induction chromatography. Trends in Biotechnology, 2002. 20(8): p. 333-337.
    連結:
  19. 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): p. 1-14.
    連結:
  20. 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): p. 99-110.
    連結:
  21. 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): p. 161-176.
    連結:
  22. 28. Tanford, C., The Hydrophobic Effect : Formation of Micelles and Biological Membranes. 1980, New York: Wiley-Interscience.
    連結:
  23. 30. Porath, J., Salt-promoted adsorption: Recent developments. Journal of Chromatography: Biomedical Applications, 1986. 376: p. 331-341.
    連結:
  24. 31. Hofstee, B.H.J., Hydrophobic affinity chromatography of proteins. Analytical Biochemistry, 1973. 52(2): p. 430-448.
    連結:
  25. 32. Shaltiel, S. and William, B.J., Hydrophobic chromatography. Methods in Enzymology, 1984. 104: p. 69-96.
    連結:
  26. 33. 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): p. 200-215.
    連結:
  27. 34. 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: p. 1-15.
    連結:
  28. 35. 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): p. 143-149.
    連結:
  29. 36. Besseling, N.A.M., Theory of Hydration Forces between Surfaces. Langmuir, 1997. 13(7): p. 2113-2122.
    連結:
  30. 37. Dill, K.A., Dominant forces in protein folding. Biochemistry, 1990. 29(31): p. 7133-7155.
    連結:
  31. 39. 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): p. 111-122.
    連結:
  32. 41. Chiu, H.-C., Lin, C.-W. and Suen, S.-Y., Isolation of lysozyme from hen egg albumen using glass fiber-based cation-exchange membranes.Joural of membrane science 290,2007.259-266.
    連結:
  33. 42. Oscarsson, S. and Porath, J., Protein chromatography with pyridine- and alkylthioether-based agarose adsorbents. Journal of Chromatography A, 1990. 499: p. 235-247.
    連結:
  34. 43. 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: p. 248.
    連結:
  35. 44. Chen, C.-I., Chen, C.-W., Huang, C.-W., Liu, Y.-C., Simultaneous purification and immobilization of penicillin G acylase using bifunctional membrane. Journal of Membrane Science, 2007. 298(1-2): p. 24-29.
    連結:
  36. 45. 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.
    連結:
  37. 46. Hu, H.-L., 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): p. 76-84.
    連結:
  38. 48. 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): p. 111-115.
    連結:
  39. 49. Melander, W.R., Rassi, Z. El 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: p. 3-27.
    連結:
  40. 50. 陳靜雯, 疏水官能基薄膜之置備及其純化盤尼西林醯胺酵素上之應用. 國立中興大學碩士學位論文, 2007.
    連結:
  41. 51. Hung, T.C., Giridhar, R., Chiou, S.H. and Wu, W.T., Binary immobilization of Candida rugosa lipase on chitosan.Joural of Molecular Catalysis B:Enzymatic 26(2003)69-78
    連結:
  42. 4. 陳東煌, 離子交換膜的應用. 化工, 1999. 46(4): p. 14-23
  43. 9. 王銀善, 疏水層析, in 生物化學技術與原理, 趙永芳, Editor. 2002, 科學出版社: 北京. p. 65.
  44. 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): p. 975-982.
  45. 18. Wang, Y., Study on Purif ication Process of Recombinant HBsAg. Chinese Journal of Biotechnology, 1999. 15: p. 263-266.
  46. 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.
  47. 25. Guerrier, L., New method for the selective capture of antibodies under physiolgical conditions. Bioseparation, 2000. 9(4): p. 211-221.
  48. 29. Creighton, T.E. and Freeman, W.E., Proteins, structure and molecular properties. 1984, New York.
  49. 38. Norde, W. and Haynes, C.A., Reversibility and the mechanism of protein adsorption. In Proteins at Interface II. Fundamentals and Applications, in ACS Symposium, T.A. Horbett and J.L. Brash, Editors. 1995, American Chemical Society: Washington, DC. p. 27-40.
  50. 40. Alkema, W.B., Hensgens, C.M., Snijder, H.J., Keizer, E., Dijksdra, B.W. and Janssen, D.B. Penicillin Acylase, mutant 2001 [cited; Available from: http://www.rcsb.org/pdb/explore.do?structureId=1JX9.
  51. 47. 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.
Times Cited
  1. 游承璋(2010)。聚偏二氟乙烯薄膜固定化脂肪分解酵素之研究。中興大學化學工程學系所學位論文。2010。1-79。