Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/91526
標題: 以矽膠固定化金屬親和吸附劑整合純化及固定化過程
Integrated enzyme purification and immobilization processes with silica-based immobilized metal affinity adsorbents
作者: Chen-Xin You
尤晨欣
關鍵字: Protein purification;Enzyme immobilization;IMAC;蛋白質純化;酵素固定化;金屬親和層析
引用: 1. H., L., et al., Molecular Cell Biology, ed. 6th. 2007, New York: W. H. Freeman. 2. Chaga, G.S., Twenty-five years of immobilized metal ion affinity chromatography: past, present and future. J. Biochem. Biophys. Methods, 2001. 49: p. 313–334. 3. Sulkowski, E., Purification of proteins by IMAC. Trends in Biotechnology, 1985. 3. 4. Gaberc-Porekar, V. and V. Menart, Perspectives of immobilized-metal affinity chromatography. J. Biochem. Biophys. Methods, 2001. 49: p. 335-360. 5. Anspach, F.B., Silica-based metal chelate affinity sorbentsI. Preparation and characterization of iminodiacetic acid affinity sorbents prepared via different immobilization techniques. Journal of Chromatography A,, 1994. 672: p. 35-49. 6. Suen∗, S.-Y., Y.-C. Liu, and C.-S. Chang, Exploiting immobilized metal affinity membranes for the isolation or purification of therapeutically relevant species. Journal of Chromatography B, 2003. 797: p. 305-319. 7. Gutiérrez, R., E.M.M.d. Valle, and M.A. Galán, Immobilized Metal Ion Affinity Chromatography: Status and Trends. Separation & Purification Reviews, 2007. 36: p. 71-111. 8. PEARSON, R.G., Hard and Soft Acids and Bases. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1983. 85: p. 22. 9. Ueda, E.K.M., P.W. Gout, and L. Morganti*, Current and prospective applications of metal ion–protein binding. Journal of Chromatography A, 2003. 988: p. 1-23. 10. Block, H., et al., Immobilized-Metal Affinity Chromatography (IMAC): A Review. Methods in Enzymology, 2009. 463. 11. Scopes, R.K., Protein Purification: Principles and Practice. Springer Science & Business Media. 1987. 12. Janson, J.-C., Protein Purification: Principles, High Resolution Methods, and Applications, ed. 3th. 2012: John Wiley & Sons. 13. Zaushitsyna, O., et al., Cryostructured and Crosslinked Viable Cells Forming Monoliths Suitable for Bioreactor Applications. Topics in Catalysis, 2013. 57: p. 336-348. 14. Börner, R.A., et al., Immobilization of Clostridium acetobutylicum DSM 792 as macroporous aggregates through cryogelation for butanol production. Process Biochemistry. Vol. 49. 2014: Elsevier. 15. Katchalski-Katzir, E., Immobilized enzymes — learning from past successes and failures. Trends in Biotechnology, 1993. 11(11): p. 471-478. 16. Babu, V.R.S., et al., Stabilization of immobilized glucose oxidase against thermal inactivation by silanization for biosensor applications. Biosensors and Bioelectronics, 2003. 19: p. 1337-1341. 17. Sheldon, R.A. and S.v. Pelt, Enzyme immobilisation in biocatalysis: why, what and how. The Royal Society of Chemistry, 2013. 42: p. 6223-6235. 18. Sheldon, R.A., Enzyme Immobilization: The Quest for Optimum Performance. Advanced Synthesis & Catalysis, 2007. 349: p. 1289-1307. 19. Avnir, D., et al., Enzymes and Other Proteins Entrapped in Sol-Gel Materials. Chemistry of Materials, 1994. 6: p. 1605-1614. 20. Cao, L., L.v. Langeny, and R.A. Sheldon, Immobilised enzymes: carrier-bound or carrier-free? Current Opinion in Biotechnology, 2003. 14: p. 387-394. 21. Hartmann, M. and D. Jung, Biocatalysis with enzymes immobilized on mesoporous hosts: the status quo and future trends. Journal of Materials Chemistry, 2009. 20: p. 844-857. 22. Mateo, C., et al., Epoxy Sepabeads: A Novel Epoxy Support for Stabilization of Industrial Enzymes via Very Intense Multipoint Covalent Attachment. Biotechnol. Prog., 2002. 18: p. 629-634. 23. Mateo, C., et al., One-Step Purification, Covalent Immobilization, and Additional Stabilization of Poly-His-Tagged Proteins Using Novel Heterofunctional Chelate-Epoxy Supports. BIOTECHNOLOGY AND BIOENGINEERING, 2001. 76. 24. Guntas, G., S.F. Mitchell, and M. Ostermeier, A Molecular Switch Created by In Vitro Recombination of Nonhomologous Genes. Chemistry & Biology, 2004. 11: p. 1483-1487. 25. Ke, W., et al., Structure of an Engineered b-Lactamase Maltose Binding Protein Fusion Protein: Insights into Heterotropic Allosteric Regulation. PLoS One, 2012. 7(6): p. e39168. 26. M.S., G., et al., Comparative studies on the isothermal characteristics of proteins adsorbed under batch equilibrium conditions to ion-exchange, immobilised metal ion affinity and dye affinity matrices with different ionic strength and temperature conditions. Journal of Chromatography A, 1997. 763: p. 79-90. 27. Ma, Z., Y. Guan, and H. Liu, Superparamagnetic silica nanoparticles with immobilized metal affinity ligands for protein adsorption. Journal of Magnetism and Magnetic Materials, 2006. 301: p. 469-477. 28. Winzerling, J.J., P. Berna, and J. Porath, How to Use Immobilized Metal Ion Affinity Chromatography. METHODS: A Companion to Methods in Enzymology, 1992. 4: p. 4-13. 29. Beilen, J.B.v. and Z. Li, Enzyme technology: an overview. Protein technologies and commercial enzymes. 2002: Elsevier Science. 30. Tural, B., et al., Purification and covalent immobilization of benzaldehyde lyase with heterofunctional chelate-epoxy modified magnetic nanoparticles and its carboligation reactivity. Journal of Molecular Catalysis B: Enzymatic, 2013. 95: p. 41-47. 31. V., S., et al., Chemistry of Aqueous Silica Nanoparticle Surfaces and the Mechanism of Selective Peptide Adsorption. Journal of the American Chemical Society, 2012. 134: p. 6244-6256. 32. 何立凡, 金屬親和吸附材於蛋白質純化及固定化之應用, in 化學工程所2002, 國立中興大學. 33. 洪?祐, 延伸臂對固定化開關蛋白之影響, in 化學工程所2013, 國立中興大學. 34. 劉育銘, 以固定化金屬親和吸附薄膜固定開關蛋白作為開發生物感測器之研究, in 化學工程所2012. 35. 葉宗翰, 蛋白質固定化用雙官能基載體之開發, in 化學工程所2014, 國立中興大學. 36. 吳宗達, 以酵素法生產海藻糖與製程純化之研究, in 化學工程所2013, 國立中興大學. 37. Ho, L.-F., et al., Integrated enzyme purification and immobilization processes with immobilized metal affinity adsorbents. Process Biochemistry, 2004. 39: p. 1573-1581.
摘要: 
這次的研究,是採用以矽膠膠體作為基材改質成雙官能基,同時具有能以 IMAC 吸附酵素末端含有 His-tag 的蛋白,以及能夠和酵素產生共價鍵鍵結的 epoxy 官能基;利用此方法,省去大量的時間及金錢成本,整合酵素純化及酵素固定化的步驟。
在此次研究結果中,得到的最適化的膠體比例為 1 : 5 之膠體,也就是銅離子的鍵結量為 88.42 μmol/g 的情況下,比起其他比例之膠體的吸附效果更好;且發現在吸附時的粗酵素液中含有 25 mM imidazole 時的純化效果比未添加時的純化效果更佳,同時也具有最佳的選擇率。
完成了以共價鍵固定之固定化酵素後進行反應 pH 、溫度及重複批次操作探討,最適化的操作條件在 pH 值為 8 ,反應溫度為 40°C ,重複操作的效果在經過 12 次的操作下,其相對活性也都落在 90 % 左右未見其顯著下降。
綜合以上實驗結果,確立了此系統整合純化及固定化的可行性,且此固定化是以共價鍵固定化,在操作上較不會產生酵素脫落的情形。

Silica-based immobilized metal affinity chromatography adsorbents with various ligand densities were prepared for the purification and immobilization of poly(His)-tagged proteins. An adsorbent with a ligand density of 88.42 μmol Cu2+/g gel exhibiting the optimal selectivity. And the crude enzyme was added 25 mM imidazole the purification better results than when not added, but also has the best selectivity.
After the completion of immobilization enzyme, investigate the reaction pH, temperature and reusability. The optimal reaction temperature and reaction pH of the immobilized enzyme were identified as 40°C and 8.0. The effect of reusability after 12 operations, the relative activity is about 90% and no significant decline.
Based on the above results, we successfully establishing that this system can be used for the direct purification and immobilization of poly(His)-tagged proteins.
URI: http://hdl.handle.net/11455/91526
其他識別: U0005-1308201517244600
Rights: 同意授權瀏覽/列印電子全文服務,2018-08-19起公開。
Appears in Collections:化學工程學系所

Files in This Item:
File SizeFormat Existing users please Login
nchu-104-7102065007-1.pdf1.82 MBAdobe PDFThis file is only available in the university internal network    Request a copy
Show full item record
 

Google ScholarTM

Check


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