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研究生:陳志義
研究生(外文):Chih-I Chen
論文名稱:製備固定化金屬親和薄膜對盤尼西林醯胺酵素之純化與固定化探討
論文名稱(外文):Preparation of the Immobilized Metal Ion Membrane for Penicillin G Acylase Purification and Immobilization
指導教授:劉永銓
指導教授(外文):Yung-Chuan Liu
學位類別:博士
校院名稱:國立中興大學
系所名稱:化學工程學系所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:73
中文關鍵詞:盤尼西林醯胺酵素固定化金屬親和薄膜最適化條件製備程序雙官能基之EPO/Cu膜再生程固定酵素膜
外文關鍵詞:Penicillin G acylaseImmobilized metal affinity membraneEnzymes purificationregeneration processimmobilized PGA membranebifunctional membrane
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盤尼西林醯胺酵素(penicillin G acylase, PGA)是一重要生化觸媒,使盤尼西林G(penicillin G, PG)進行水解反應生成六青黴素酸(6-aminopenicillanic acid, 6-APA),它和一些有機物以有機合成反應形成β-lactam抗生素。在全球巿場,每年有數千噸β-lactam抗生素需求量應用於治療方面。因此製備出高活性與穩定性之PGA對於6-APA生產是很重要。近年來純化技術發展,固定化金屬親和薄膜(immobilized metal affinity membranes, IMAM)己被應用於酵素純化領域,因為它較一般填充床系統無顆粒內部擴散問題,且具有短軸擴散途徑、壓力降低和易規格化等優點,因此完整探討最適化之IMAM製備程序和再生程序以得到更佳IMAM之功能是有必要性。夲研究共分三個部份。
第一部份針對製備IMAM具高量金屬離子和蛋白質吸附能力之方法。NaOH濃度是再生纖維素膜 (RC膜) 與Epichlorohydrin (EPI) 之間接合反應的最重要關鍵步驟,在最適化反應條件: 一片RC膜浸泡於20 ml、 1.4 M NaOH及5 ml EPI溶液,以24 °C、150 rpm 反應14 h進行製備。製備完成之IMAM膜有高吸附75.5 ± 0.25 μmol Cu2+/disc 、1044 μg lysozyme/cm2 and 1.8 U PGA/disc之量,這些值高於相關文献[1]之研究結果。使用10片IMAM裝置於流動式層析之筒匣內,在18℃下,以10 mM、 pH 6 之PB buffer為負載液,1.5 M NH4Cl為洗滌液,以流動式層析進行PGA純化試驗,有97% PGA 回收率和21.31 倍純化倍率之結果。
第二部份針對雙官能基之EPO/Cu膜 (epoxy and immobilized copper ion membrane,簡稱EPO/Cu)對PGA 純化與固定化之吸附機制探討。Iminodiacetic acid disodium (IDA)濃度是製備雙官能基之EPO/Cu膜的最重要關鍵地方,當使用0.05M IDA製備之EPO/Cu膜,僅螯合29.9
μmol Cu2+/disc, 是螯合飽和量之58% ,但此EPO/Cu膜歷時2個月26次純化與固定化測試,有96.3% PGA固定在EPO/Cu膜。
第三部份針對固定酵素之IPM膜(the immobilized PGA membrane ,簡稱IPM)之再生方法。由製備完成之IMAM膜吸附PGA成IPM膜,探討IPM膜之PGA穩定性與再生方法。IPM膜放入4 ºC之DI water內,經10天連續測試後IPM之PGA活性剩下19%,同樣地,若放入10mM PB buffer (含0.1% NaN3, pH8) 經10天連續測試後,IPM之PGA活性維持在99%以上,非常穩定。在再生方面,使用100mM EDTA當刮除液,經連續4次再生步驟後,IPM固定PGA能力只剩下52%,同樣地,若使用各刮除溶液浸泡,如100mM EDTA (300mM NaCl, 20mM PB, pH8)浸泡30 min, 接著浸於25ml、0.5 M HCl與25ml、 0.5 M NaOH各10 min等為再生程序,經連續5次再生程序後, IPM固定PGA活性仍維持在99%以上,非常穩定。
Penicillin G acylase (PGA) is an important bioenzyme to hydrolyze penicillin G into 6-aminopenicillanic acid (6-APA) which could react with some organic chemicals by organic synthetic process to form β-lactam antibiotics. The global market demand of β-lactam antibiotics reaches thousand of tons annually for medical treatment. However, the preparation of highly active and stable PGA is thereby critical for 6-APA production. Among the recently developed purification techniques, immobilized metal ion affinity membrane (IMAM) has been widely applied in the enzyme purification processes with advantages such as no intra-particle diffusion, short axial-diffusion path, low pressure drop and easier scale up, which limited in conventional packed-column systems. To achieve better IMAM performance, a thorough study for optimizing the IMAM-preparation procedures and regeneration process is required. In this paper, it contains three parts.
In the first part, an approach to prepare IMAM with high adsorption capacities of metal ions and protein was developed. NaOH concentration is found to be the most influential one for the reaction of coupling epichlorohydrin (EPI) to the regenerated cellulose membrane (RC membrane). According to the criterion, the optimal reaction conditions were found as follows: a RC membrane immersed in 20 ml, 1.4 M NaOH, 5 ml EPI and operated at 24 °C, 150 rpm for 14 h. Under this condition, the copper ions and PGA in IMAM were significantly increased to 75.5 ± 0.25 μmol/disc and 1.8 U/disc respectively. The adsorption amount for lysozyme on the prepared IMAM reached 1044 μg/cm2, which was the highest in the literature. We could get the result of 97.1% recovery for PGA purification, which is 21.3-fold than before, by loading 10 pieces of IMAM into the cartridge, conducting the flow chromatography at 18 ºC with 10 mM, using pH 6 sodium phosphate buffer as loading buffer and 1.5 M NH4Cl as elution buffer, a 21.3-fold purification with 97.1% recovery for PGA purification was obtained.
In the second part, the bifunctional membrane (epoxy and immobilized copper ion) was constructed to study the effect on PGA purification and immobilization. It is found that iminodiacetic acid disodium (IDA) concentration has the critically effect on the bifunctional group ratio in the membrane. When IDA concentration was 0.050 M, the chelated copper ion would reach 29.9 μmol/disc, nearly 58% of its saturation. After purification and immobilization cycling process 26 times within two month, 96.3% of the PGA activity could be still retained.
In the third part, the stability and regeneration of immobilized PGA membrane (IPM) by using pre-IMAM to immobilize PGA reversibly is investigated. IPM was stored in deionized water (DI) at 4 ºC, its residual activity was 19 % left of the original one after 10 days. However, when stored in 10mM phosphate buffer (PB, pH8) with 0.1% NaN3, the IPM can be more stable which retains 99% of its activity after 10 days testing. In regeneration, the re-immobilization capacity for PGA is only 52% left of the original one after 4 times regeneration by using 100mM EDTA as the stripping buffer. When the regeneration process was modified by immersing in 100mM EDTA (300mM NaCl, 20mM PB, pH8) for 30 min, in 25ml, 0.5 M HCl for 10 min, then rinsed twice with DI water and finally immersed in 25ml, 0.5 M NaOH for 10 min. The resulting IMAM can reserve 99% PGA immobilized activity even after 5-times regeneration.
目 錄
摘 要 I
Abstract III
目 錄 V
第一章 緒 論 1
1-1 前言 1
1-2 研究動機與目標 2
第二章 文獻回顧 4
2-1盤尼西林醯胺酵素 4
2-2固定化金屬親和層析法 6
2-3薄膜應用於金屬親和層析法 12
2-4酵素固定在載體 13
2-5可逆固定化技術 15
第三章 實驗材料與方法 16
3-1 實驗材料與設備 16
3-1-1材料與藥品 16
3-1-2儀器: 17
3-2 實驗方法 18
3-2-1 PGA酵素液的製備 18
3-2-2 蛋白質濃度的量測 18
3-2-3 PGA活性的量測 19
3-2-4 環氧基之量測 20
3-2-5 EPI 與 IDA在RC膜之量測 20
3-2-6 IMAM膜張力之量測 21
3-2-7 IMAM膜與 EPO/Cu膜之製備步驟 21
3-2-8 銅離子量的量測 22
3-2-9 製備吸附PGA或lysozyme之IMAM膜 22
3-2-10 流動式層析裝置純化PGA 23
3-2-11 膜吸附PGA之活性測量 23
3-2-12 IPM膜之製備及再生程序 25
第四章 製備金屬離子與蛋白質高吸收效率之IMAM膜 27
4-1前言 27
4-2 結果與討論 27
4-2-1 NaOH濃度對製備IMAM之影響 27
4-2-2 EPI接合反應之最適化條件探討 28
4-2-3 IDA濃度與銅離子量之影響 29
4-2-4反應生成轉化率之探討 29
4-2-5 IMAM功能之比較 30
4-2-6 以IMAM膜流動式層析程序純化PGA 30
4-3結論 31
第五章 使用雙功能之EPO/Cu膜同時純化與固定化PGA 41
5-1前言 41
5-2 結果與討論 41
5-2-1 双官能基EPO/Cu 膜之製備 41
5-2-2 雙官能基EPO/Cu 膜對PGA之純化與固定化 42
5-2-3 雙官能基EPO/Cu 膜固定PGA之穩定性與效率 42
5-3 結論 44
第六章 IPM膜可逆固定化酵素方法 52
6-1 前言 52
6-2 結果與討論 52
6-2-1 探討IMAM固定PGA量之變化 52
6-2-2 IPM膜之穩定性探討 52
6-2-3 使用Imidazole或 EDTA對IPM再生程序之影響 53
6-2-4使用SEM分析IPM膜表面狀態 53
6-2-5 IPM膜之再生程序 54
6-3結論 54
第七章 結論及未來展望 60
7-1結論 60
7-2未來展望 61
參考文獻 62
參考文獻
[1]H. L. Hu, M. Y. Wang, C. H. Chung, S. Y. Suen, Purification of VP3 protein of infectious bursal disease virus using nickel ion-immobilized regenerated cellulose-based membranes, J Chromatogr B (2006) 840 76-84.
[2]P. Knight, Downstream Processing, Nat Biotechnol (1989) 7 777-82.
[3]R. Ghosh, Protein separation using membrane chromatography: opportunities and challenges, J Chromatogr A (2002) 952 13-27.
[4]S. Y. Suen, Y. C. Liu, C. S. Chang, Exploiting immobilized metal affinity membranes for the isolation or purification of therapeutically relevant species, J Chromatogr B (2003) 797 305-19.
[5]R. Freitag, H. Splitt, O.-W. Reif, Controlled mixed-mode interaction chromatography on membrane adsorbers, J Chromatogr A (1996) 728 129-37.
[6]V. Fitton, N. Verdoni, J. Sanchez, X. Santarelli, Penicillin acylase purification with the aid of pseudo-affinity chromatography, J Biochem Biophys Methods (2001) 49 553-60.
[7]S. Senel, R. Say, Y. Arica, A. Denizli, Zinc ion-promoted adsorption of lysozyme to Cibacron Blue F3GA-attached microporous polyamide hollow-fiber membranes, Colloids surface A (2001) 182 161-73.
[8]J. Crawford, S. Ramakrishnan, P. Periera, S. Gardner, M. Coleman, R. Beitle, Immobilized metal affinity membrane separation: characteristics of two materials of differing preparation chemistries, Separation Sci. Technol. (1999) 34 2793-802.
[9]R. Kumar, R. Prasad, Purification and characterization of a major zinc binding protein from renal brush border membrane of rat, Biochimica et Biophysica Acta ( 1999) 1419 23-32.
[10]C. Y. Wu, S. Y. Suen, S. C. Chen, J. H. Tzeng, Analysis of protein adsorption on regenerated cellulose-based immobilized metal affinity membranes, J Chromatogr A (2003) 996 53-70.
[11]B. M, P. J, Immobilized metal ion affinity chromatography. Effect of solute structure, ligand density and salt concentration on the retention of peptides., J Chromatogr A (1990) 516 333-54.
[12]F. B. Anspach, Silia-based metal chelate affinity sorbents I. Preparation and characterization of iminodiacetic acid affinity sorbents prepared via different immobilization techniques, J Chromatogr A (1994) 672 35-49.
[13]陳光宇, 大腸桿菌醱酵生產盤尼西林G醯胺酵素代謝工程之研究, 國立中興大學化學工程研究所碩士論文 (1999)
[14]黃宏彰, 高細胞密度醱酵培養基因重組大腸桿菌以生產盤尼西林醯胺酵素, 國立中興大學化學工程研究所碩士論文 (2002)
[15]Y. C. Liu, C. C. ChangChien, S. Y. Suen, Purification of penicillin G acylase using immobilized metal affinity membranes, J Chromatogr B (2003) 794 67-76.
[16]G. Schumacher, D. Sizmann, H. Haug, P. Buckel, A. Böck, Penicillin acylase from E. coli: unique gene-protein relation., Nucleic Acids Res (1986) 14 5713-27.
[17]蔡嘉寅, 陳益明, 生物化學工程. 2002: 國立台灣大學生物技術研究中心.
[18]E. Sulkowski, Purification of proteins by IMAC, Trends Biotechnol. (1985) 3 1-7.
[19]E. S. Hemdan, J. Porath, Development of immobilized metal affinity chromatography : II. Interaction of amino acids with immobilized nickel iminodiacetate, J Chromatogr A (1985) 323 255-64.
[20]R. D. Johnson, F. H. Arnold, Multiponit binding and heterogeneity in immobilized metal affinity chromatography, Biotechnol Bioeng (1995) 48 437-43.
[21]N. Kubota, Y. Nakagawa, Y. Eguchi, Recovery of serum proteins using cellulosic affinity membrane modified by Immobilization of Cu2+ Ion, J Appl Polym Sci Symp (1996) 62 1153-60.
[22]L. Yang., L. Jia, H. Zou, D. Zhou, Y. Zhang, Immobilized metal affinity composite membrane based on cellulose for separation of biopolymers, Science in China. Series B, Chemistry, life sciences & earth sciences (1998) 41 596-605.
[23]T. C. Beeskow, W. Kusharyoto, K. H. Kroner, W. D. Deckwer, F. B. Anspach, Surface modification of microporous polyamide membranes with hydroxyethyl cellulose and their application as affinity membranes, J Chromatogr A (1995) 715 49-65.
[24]M. Grasselli, A. A. N. del Canizo, S. A. Camperi, F. J. Wolman, E. E. Smolko, O. Cascone, Immobilized metal ion affinity hollow-fiber membranes obtained by the direct grafting technique, Radiat. Phys. Chem. (1999) 55 203-08.
[25]O.-W. Reif, V. Nier, U. Bahr, R. Freitag, Immobilized metal affinity membrane adsorbers as stationary phases for metal interaction protein separation, J Chromatogr A (1994) 664 13-25.
[26]C.-M. Zhang, S. A. Reslewic, C. E. Glatz, Suitability of immobilized metal affinity chromatography for protein purification from canola, Biotechnol Bioeng (1999) 68 52-58.
[27]S. Gibert, N. Bakalara, X. Santarelli, Three-step chromatographic purification procedure for the production of a His-tag recombinant kinesin overexpressed in E . coli, J Chromatogr B (2000) 737 143-50.
[28]J. Sanchez, N. Verdoni, V. Fitton, X. Santarelli, Efficient two-step chromatographic purification of penicillin acylase from clarified Escherichia coli ultrasonic homogenate J Chromatogr B (2001) 753 45-50.
[29]H. Zou, Q. Luo, D. Zhou, Affinity membrane chromatography for the analysis and purification of proteons, J Biochem Biophys Methods (2001) 49 199-240.
[30]何立凡, 金屬親和吸附材於蛋白質純化之應用, in 國立中興大學化學工程研究所碩士論文. 2002.
[31]Z. E. Rassi, C. Horvath, Metal chelate-interaction chromatography of proteins with iminodiacetic acid-bonded stationary phases on silica support, J Chromatogr (1986) 359 241-53.
[32]L. A, S.-N. C, L. KP, M. KG, B.-H. TC, Divinylsulphone-activated agarose. Formation of stable and non-leaking affinity matrices by immobilization of immunoglobulins and other proteins, J Chromatogr. (1986) 376 299-305.
[33]J. Liesiene, K. Racaityte, M. Morkeviciene, P. Valancius, B. Bumelis, Immobilized metal affinity chromatography of human growth hormone Effect of ligand density, J Chromatogr A (1997) 764 27-33.
[34]S. Vancan, E. A. Miranda, S. M. A. Bueno, IMAC of human IgG: studies with IDA-immobilized copper, nickel, zinc, and cobalt ions and different buffer systems, Proc Biochem (2002) 37 573-79.
[35]P. Armisen, C. Mateo, E. Cortes, J. L. Barredo, F. Salto, B. Diez, L. Rodes, J. L. Garc, R. Fernandez-Lafuente, J. M. Guisan, Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports, J Chromatogr A (1999) 848 61-70.
[36]F. H. Arnold, Metal-Affinity Separations: A New Dimension in Protein Processing, Nat Biotechnol (1991) 9 151-56.
[37]E. K. M. Ueda, P. W. Gout, L. Morganti, Current and prospective application of metal ion-protein binding, J Chromatogr A (2003) 988 1-23.
[38]V. Gaberc-Porekar, V. Menart, Reveiw perspectives of immobilized-metal affinity chromatography, J. Biochem. Biophys. Methods (2001) 49 335-60.
[39]G. S. Chaga, Review twenty-five years of immobilized metal ion affinity chromatography: past, present and future, J. Biochem. Biophys. Methods (2001) 49 313-34.
[40]V. Gaberc-Porekar, V. Menart, Perspectives of immobilized-metal affinity chromatography, J Biochem Biophys Methods (2001) 49 335-60.
[41]G. S. Chaga, B. Ersson, J. O. Porath, Isolation of calcium-binding proteins on selective adsorbents application to purification of bovine calmodulin, J. Chromatogr. A (1996) 732 261-69.
[42]J. Porath, B. Olin, B. Granstrand, Immobilized-metal affinity chromatography of serum proteins on gel-immobilized group IIIA metal ions, Arch. Biochem. Biophys. (1983) 225 543-47.
[43]G. Chaga, J. Hopp, P. Nelson, Immobilized metal ion affinity chromatography on Co2+-carboxymethylaspartate-agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase from chicken breast muscle, Biotechnol. Appl. Biochem. (1999) 29 19-24.
[44]C. Mateo, G. Fernandez-Lorente, E. Cortes, J. L. Garcia, R. Fernandez-Lafuente, J. M. Guisan, One-step purification, covalent immobilization, and additional stabilization of poly-his-tagged proteins using novel heterofunctional chelate-epoxy supports, Biotechnol Bioeng (2001) 76 269-76.
[45]C. Mateo, G. Fernandez-Lorente, B. C. C. Pessela, A. Vian, A. V. Carrascosa, J. L. Garcia, R. Fernandez-Lafuente, J. M. Guisan, Affinity chromatography of polyhistidine tagged enzymes new dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions, J Chromatogr A (2001) 915 97-106.
[46]Y.-C. Liu, S.-Y. Suen, C.-W. Huang, C.-C. ChangChien, Effects of spacer arm on penicillin G acylase purification using immobilized metal affinity membranes, J Membr Sci (2005) 251 201-07.
[47]V. Fitton, X. Santarelli, Evaluation of immobilized metal affinity chromatography for purification of penicillin acylase, J Chromatogr B (2001) 754 135-40.
[48]A. Kotha, L. Selvaraj, C. R. Rajan, S. Ponrathnam, K. K. Kumar, G. R. Ambekar, J. G. Shewale, Adsorption and expression of penicillin G acylase immobilized onto methacrylate polymers generated with varying pore generating solvent volume Appl Biochem Biotechnol (1991) 30 297-302.
[49]A. Naidja, P. M. Huang, J.-M. Bollag, Activity of tyrosinase immobilized on hydroxyaluminum-montmorillonite complexes Journal of Molecular Catalysis A: Chemical (1997) 115 305-16.
[50]J. He, X. Li, D. G. Evans, X. Duan, C. Li, A new support for the immobilization of penicillin acylase, Journal of Molecular Catalysis B: Enzymatic (2000) 11 45-53.
[51]田蔚城, 生物技術的發展與應用, 九州圖書文物有限公司 (1998)
[52]陳國誠, 生物固定化技術與產業應用, 茂昌圖書有限公司 (2000)
[53]潘建亮, 固定化盤尼西林去醯基酵素反應動力學建模及其兩水相系統分離反應之探討, 國立成功大學化學工程研究所博士論文 (2005)
[54]Y. K, H. S, Gradient and isocratic high-performance liquid chromatography of proteins on a new agarose-based anion exchanger, J Chromatogr. (1987) 385 87-98.
[55]M. VV, Mechanism-based strategies for protein thermostabilization, Trends Biotechnol. (1993) 11 88-95.
[56]B. R. M., G. J. M., Stabilisation of enzymes by multipoint covalent attachment to agarose-aldehyde gels. Borohydride reduction of trypsin-agarose derivatives, Enzyme and microbial technology (1989) 11 360-66.
[57]E. Katchalski-Katzir, Immobilized enzymes - learning from past successes and failures, Trends in Biotechnology (1993) 11 471-78.
[58]A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts, B. Witholt, Industrial biocatalysis today and tomorrow, Nature (2001) 409 258-68.
[59]J. Turkova, Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function, J Chromatogr B (1999) 722 11-31.
[60]C. Mateo, O. Abian, R. Fernandez-Lafuente, J. M. Guisan, Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support-polyethylenimine composites, Biotechnol Bioeng (2000) 68 95-105.
[61]B. C. C. Pessela, M. Fuentes, C. Mateo, R. Munilla, A. V. Carrascosa, R. Fernandez-Lafuente, J. M. Guisan, Purification and very strong reversible immobilization of large proteins on anionic exchangers by controlling the support and the immobilization conditions Enzyme Microb Technol (2006) 39 909-15.
[62]M. Y. Arıca, G. Bayramoglu, Reversible immobilization of tyrosinase onto polyethyleneimine-grafted and Cu(II) chelated poly(HEMA-co-GMA) reactive membranes, Journal of Molecular Catalysis B: Enzymatic (2004) 27 255-65.
[63]M. M. Bradford, , A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem (1976) 72 248-54.
[64]S. Kroll, L. Meyer, A. M. Graf, S. Beutel, S. D¨oring, U. Klaus, T. Scheper, Heterogeneous surface modification of hollow fiber membranes for use in micro-reactor systems, J Memb Sci (2007) 299 181-89.
[65]R. D. Johnson, R. J. Todd, F. H. Arnold, Multipoint binding in metal-affinity chromatography II. Effect of pH and imidazole on chromatographic retention of engineered histidine-containing cytochromes J Chromatogr A (1996) 725 225-35.
[66]K. Rodemann, E. Staude, Synthesis and characterization of affinity membranes made from polysulfone, J Memb Sci (1994) 88 271-78.
[67]C. Mateo, O. Abian, R. Fernandez-Lafuente, J. M. Guisan, Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment, Enzyme Microbial Technol. (2000) 26 509-15.
[68]C. Mateo, O. Abian, J. M. Guisan, Multifunctional Epoxy Supports: A New Tool To Improve the Covalent Immobilization of Proteins. The Promotion of Physical Adsorptions of Proteins on the Supports before Their Covalent Linkage, Biomacromolecules (2000) 1 739-45.
[69]B. C. C. Pessela, C. Mateo, A. V. Carrascosa, A. Vian, J. L. Garcia, G. Rivas, C. Alfonso, J. M. Guisan, R. Fernandez-Lafuente, One-Step Purification, Covalent Immobilization, and Additional Stabilization of a Thermophilic Poly-His-Tagged β-Galactosidase from Thermus sp. Strain T2 by using Novel Heterofunctional Chelate-Epoxy Sepabeads, Biomacromolecules (2003) 4 107-13.
[70]C. Mateo, R. Torres, G. Fernandez-Lorente, C. Ortiz, M. Fuentes, A. Hidalgo, F. Lopez-Gallego, O. Abian, J. M. Palomo, L. Betancor, B. C. C. Pessela, J. M. Guisan, R. Fernandez-Lafuente, Epoxy-Amino Groups: A New Tool for Improved Immobilization of Proteins by the Epoxy Method, Biomacromolecules (2003) 4 772-77.
[71]C. Mateo, O. Abian, G. Fernandez-Lorente, J. Pedroche, R. Fernandez-Lafuente, J. M. Guisan, Epoxy sepabeads: a novel epoxy support for stabilization of industrial enzymes via very intense multiponit covalent attachment, Biotechnol. Prog. (2002) 18 629-34.
[72]D. Bianchi, P. Golini, R. Bortolo, P. Cesti, Immobilization of penicillin G acylase on aminoalkylated polyacrylic supports, Enzyme Microb Technol (1996) 18 592-96.
[73]J. Sanchez, N. Verdoni, V. Fitton, X. Santarelli, Efficient two-step chromatographic purification of penicillin acylase from clarified Escherichia coli ultrasonic homogenate, J Chromatogr B (2001) 753 45-50.
[74]L. Yang, L. Jia, H. Zou, Y. Zhang, Immobilized iminodiacetic acid (IDA)-type Cu2+-chelatingmembrane afnity chromatography for purication of bovine liver catalase, Biomed Chromatogr (1999) 13 229-34.
[75]L. C. L. Aquino, H. R. T. Sousa, E. A. Mirand, L. Vilela, S. M. A. Bueno, Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin, J Chromatogr B (2006) 834 68–76.
[76]S. Gisele, F. P. A. Elisabeth, M. S. C. T.Wirla, B. R.Mariana, E. M.verson, M. A. BSonia, Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G1 monoclonal antibody, J Chromatogr B (2005) 816 259–68.
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