Please use this identifier to cite or link to this item:
Investigation on the stability and regeneration of penicillin G acylase immobilized in immobilized metal affinity membrane
|關鍵字:||immobilized metal affinity membrane;固定化金屬親和層析薄膜;penicillin G acylase;regeneration;purification;盤尼西林醯胺酵素;酵素固定化;酵素純化;再生||出版社:||化學工程學系所||引用:|| C. Charcosset, Purification of proteins by membrane chromatography. Journal of Chemical Technology and Biotechnology, 1998. 71: p. 95.  F.H. Arnold, Metal-affinity separations: a new dimension in protein processing. Bio/Technology 1991. 9: p. 151.  V. Fitton, Evaluation of immobilized metal affinity chromatography for purification of penicillin acylase. Journal of Chromatography B, 2001. 754: p. 135.  V. Gaberc-Porekar and V. Menart, Perspectives of immobilized-metal affinity chromatography. Journal of Biochemical and Biophysical Methods, 2001. 49: p. 335.  G.S. Chaga, Twenty-five years of immobilized metal ion affinity chromatography: past, present and future. Journal of Biochemical and Biophysical Methods 2001. 49: p. 313.  J. Porath, Immobilizded metal Ion affinity chromatography. Protein Expression and Purification, 1992. 3: p. 263.  S.-Y. Suen, 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.  P.R. Hari, W. Paul and C.P. Sharma, Adsorption of human IgG on Cu2+-immobilized cellulose affinity membrane: Preliminary study. Journal of Biomedical Materials Research, 2000. 50: p. 110.  S. Senel, R. Say, Y. Arica and A. Denizli, Zinc ion-promoted adsorption of lysozyme to Cibacron Blue F3GA-attached microporous polyamide hollow-fiber membranes. Colloids Surface A, 2001. 182: p. 161.  J. Crawford, S. Ramakrishnan, P. Periera, S. Gardner, M. Coleman and R. Beitle, Immobilized metal affinity membrane separation: characteristics of two materials of differing preparation chemistries. Separation and Purification Technology, 1999. 34: p. 2793.  R. Kumar and R. Prasad, Purification and characterization of a major zinc binding protein from renal brush border membrane of rat. Biochimica et Biophysica Acta 1999. 1419: p. 23.  R.R. Begum, R.J. Newbold and D. Whitford, Purification of the membrane binding domain of cytochrome b5 by immobilized nickel chelate chromatography. Journal of Chromatography B, 2000. 737: p. 119.  H.-L. Hu, M.-Y. Wang, C.-H. Chung and S.-Y. Suen, Purification of VP3 Protein of Infectious Bursal Disease Virus Using Immobilized Nickel Ion Regenerated Cellulose-Based Membranes. Journal of Chromatography B, 2006. 840: p. 76.  C.-S. Chen, S.-Y. Suen, S.-Y. Lai, G. R.-L. Chang, T.-C. Lu, M.-S. Lee and M.-Y. Wang, Purification of Capsid-like Particles of Infectious Bursal Disease Virus (IBDV) VP2 Expressed in E. coli with a Metal-ion Affinity Membrane System. Journal of Virological Methods, 2005. 130: p. 51.  C.-Y. Wu, S.-Y. Suen, S.-C. Chen and J.-H. Tzeng, Analysis of Protein Adsorption on Regenerated Cellulose-based Immobilized Copper Ion Affinity Membranes. Journal of Chromatography A, 2003. 996: p. 53.  F.B. Anspach, Silia-based metal chelate affinity sorbents I. Preparation and characterization of iminodiacetic acid affinity sorbents prepared via different immobilization techniques. Journal of Chromatography A, 1994. 672: p. 35.  M. Belew, Immobilized metal ion affinity chromatography effect of solute structure, ligand density and salt concentration on the retentation of peptides. Journal of Chromatography A, 1990. 516: p. 333.  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.  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.  V. Grazu, O. Abian, C. Mateo, F. Batista-Viera, R. Fernandez-Lafuente and J.M. Guisan, Novel bifunctional epoxy/thiol-reactive support to immobilize thiol containing proteins by the epoxy chemistry. Biomacromolecules, 2003. 4: p. 1495.  C. Mateo, O. Abian, G. Fernandez-Lorente, J. Pedroche, R. Fernandez-Lafuente, J.M. Guisan, A. Tam and M. Daminati, Epoxy sepabeads: a novel epoxy support for stabilization of industrial enzymes via very intense multipoint covalent attachment. Biotechnology Progress 2002. 18: p. 629.  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. Pessela, J.M. Guisan and R. Fernandez-Lafuente, Epoxy-amino groups: a new tool for improved immobilization of proteins by the epoxy method. Biomacromolecules, 2003. 4: p. 772.  B.C. Pessela, C. Mateo, A.V. Carrascosa, A. Vian, J.L. Garcia, G. Rivas, C. Alfonso, J.M. Guisan and R. Fernandez-Lafuente, One-step purification, covalent immobilization, and additional stabilization of a thermophilic poly-His-tagged beta-galactosidase from Thermus sp. strain T2 by using novel heterofunctional chelate-epoxy Sepabeads. Biomacromolecules, 2003. 4: p. 107.  B.C. Pessela, C. Mateo, M. Fuentes, A. Vian, J.L. Garcia, A.V. Carrascosa, J.M. Guisan and R. Fernandez-Lafuente, Stabilization of a multimeric beta-galactosidase from Thermus sp. strain T2 by immobilization on novel heterofunctional epoxy supports plus aldehyde-dextran cross-linking. Biotechnology Progress, 2004. 20: p. 388.  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.  R.R. Yocum, J.R. Rasmussen and J.L. Strominger, The mechanism of action of penicillin. Journal of Biological Chemistry 1980. 255: p. 3977.  E. Sulkowski, Purification of proteins by IMAC. Trends in Biotechnology, 1985. 3: p. 1.  E.S. Hemdan and J. Porath, Development of immobilized metal affinity chromatography II. Interaction of amino acids with immobilized nickel iminodiaceta. Journal of Chromatography, 1985. 323: p. 255.  W.Q.B. Shixiang and Y. Ruhua, Progress of Immobilized Metal-chelate Affinity Chromatography. South China University of Technology, Guangzhou Chinese Academy of Tropical Agricultural Sciences, Haikou, 1995.  H. Zou, Q. Luo and D. Zhou, Affinity membrane chromatography for the analysis and purification of proteons. Journal of Biochemical and Biophysical Methods, 2001. 49: p. 199.  何立凡, 金屬親和吸附材於蛋白質純化之應用. 國立中興大學化學工程研究所碩士論文, 2002.  A. Lihme, C. Schafer-Nielsen, K.P. Larsen, K.G. Muller and T.C. Bog-Hansen, Divinylsulphone-activated agarose formation of stable and non-leaking affinity matrices by immobilization of immunoglobulins and other proteins. Journal of Chromatography, 1986. 376: p. 299.  S. Vancan, E.A. Miranda and S.M.A. Bueno, IMAC of human IgG: studies with IDA-immobilized copper,nickel, zinc, and cobalt ions and different buffer syste. Process Biochemistry, 2002. 37: p. 573.  P. Armisen, C. Mateo, E. Cortes, J.L. Barredo, F. Salto, B. Diez, L. Rodes, J.L. Garc, R. Fernandez-Lafuente and J.M. Guisan, Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports. Journal of Chromatography A, 1999. 848: p. 61.  E.S. Hemdan and J. Porath, Development of immobilized metal affinity chromatography I. Comparision of two iminodiacetate gels. Journal of Chromatography, 1985. 323: p. 247.  J. Liesiene, K. Racaityte, M. Morkeviciene, P. Valancius and V. Bumelis, Immobilized metal affinity chromatography of human growth hormone Effect of ligand density1. Journal of Chromatography A 1997. 764: p. 27.  L. Gelunaite, V. Luksa, O. Sudziuviene, V. Bumelis and H. Pesliakas, Chelated mercury as a ligand in immobilized metal ion affinity chromatography of proteins. Journal of Chromatography A, 2000. 904: p. 131.  E.K.M. Ueda, P.W. Gout and L. Morganti, Current and prospective application of metal ion-protein binding. Journal of Chromatography A, 2003. 988: p. 1.  G.S. Chaga, B. Ersson and J.O. Porath, Isolation of calcium-binding proteins on selective adsorbents application to purification of bovine calmodulin. Journal of Chromatography A, 1996. 732: p. 261.  J. Porath, B. Olin and B. Granstrand, Immobilized-metal affinity chromatography of serum proteins on gel-immobilized group III A metal ions. Archives of Biochemistry and Biophysics, 1983. 225: p. 543.  X. Santarelli, N. Verdoni, V. Fitton and Sanchez, Efficient two-step chromatographic purification of penicillin acylase from clarified Escherichia coli ultrasonic homogenate. Journal of Chromatography B, 2001. 753: p. 45.  S. Gibert, N. Bakalara and X. Santarelli, Three-step chromatographic purification procedure for the production of a His-tag recombinant kinesin overexpressed in E . coli. Journal of Chromatography B, 2000. 737: p. 143.  C.-M. Zhang, S.A. Reslewic and C.E. Glatz, Suitability of immobilized metal affinity chromatography for protein purification from canola. Biotechnology and Bioengineering, 1999. 68: p. 52.  A.C.B.d. Genaro and R.E. Tamagawa, Recovery and purification of aprotinin from industrial insulin processing effluent by immobilized chymotrypsin and negative IMAC chromatographies. Process Biochemistry 2002. 37: p. 1413.  A.E. Price, K.B. Logvinenko, E.A. Higgins, E.S. Cole and S M Richards, Studies on the microheterogeneity and in vitro activity of glycosylated and nonglycosylated recombinant human prolactin separated using a novel purification process. Endocrinology, 1995. 136: p. 4827.  W. Jiang and M.T.W. Hearn, Protein interaction with immobilized metal ion affinity ligands under high ionic strength conditions. Analytical Biochemistry 1996. 242: p. 45.  Z.E. Rassi and C. Horvath, Metal chelate-interaction chromatography of proteins with iminodiacetic acid-bonded stationary phases on silica support. Journal of Chromatography, 1986. 359: p. 241.  G.M.S. Finette, Q.-M. Mao and M.T.W. Hearn, Comparative studies on the isothermal characteristics of proteins adsorbed under batch equilibrium conditions to ion-exchange, immobilized metal ion affinity and dye affinity matrices with different ionic strength and temperature conditions. Journal of Chromatography A 1997. 763: p. 71.  T.C. Beeskow, W. Kusharyoto, F.B. Anspach, K.H. Kroner and W.-D. Deckwer, Surface modification of microporous polyamide membranes with hydroxyethyl cellulose and their application as affinity membranes. Journal of Chromatography A, 1995. 715: p. 49.  K. Rodemann and E. Staude, Polysulfone affinity membranes for the treatment of amino acids mixtures. Biotechnology and Bioengineering, 1995. 46: p. 503.  M. Grasselli, S.A. Camperi, F.J. Wolman, E.E. Smolko and O. Cascone, Immobilized metal ion affinity hollow-fiber membranes obtained by the direct grafting technique. International Journal of Radiation Biology and Related Studies in Physics, Chemistry, and Medicine, 1999. 55: p. 203.  O.-W. Reif, V. Nier, U. Bahr and R. Freitag, Immobilized metal affinity membrane adsorbers as stationary phases for metal interaction protein separation. Journal of Chromatography A, 1994. 664: p. 13.  Q. Zeng, J. Xu, R. Fu and Q. Ye, Functional polymer affinity matrix for purifying hexahistidine-tagged recombinant protein. Journal of Chromatography A, 2001. 921: p. 197.  Q. Luo, H. Zou, X. Xiao, Z. Guo, L. Kong and X. Mao, Chromatographic separation of proteins on metal immobilized iminodiacetic acid-bound molded monolithic rods of macroporous poly(glycidyl methacrylate O-ethylene dimethacrylat). Journal of Chromatography A, 2001. 926: p. 255.  L. Yang, L. Jia, H. Zou and Y. Zhang, Immobilized iminodiacetic acid (IDA)-type Cu2+-chelatingmembrane afnity chromatography for purication ofbovine liver catalase. Biomedical Chromatography 1999. 13: p. 229.  N. Kubota, Y. Nakagawa and Y. Eguchi, Recovery of serum proteins using cellulosic affinity membrane modified by Immobilization of Cu2+ Ion. Journal of Applied Polymer Science, 1996. 62: p. 1153.  G. Chaga, J. Hopp and 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. Applied Biochemistry and Biotechnology, 1999. 29: p. 19.  R.H. Clemmitt and H.A. Chase, Facilitated Downstream Processing of a Histidine-Tagged Protein from Unclarified E. coli Homogenates Using Immobilized Metal Affinity Expanded-Bed Adsorption. Biotechnology and Bioengineering, 2000. 67: p. 206.  U. Sidenius, O. Farver, O. Jons and B. Gammelgaard, Comparison of Different Transition Metal Ions for Immobilized Metal Affinity Chromatography of Selenoprotein P from Human Plasma. Journal of Chromatography B, 1999. 735: p. 85.  C. Mateo, G. Fernandez-Lorente, E. Cortes, J.L. Garcia, R. Fernandez-Lafuente and J.M. Guisan, One Step Purification, Covalent Immobilization, and Additional Stabilization of Poly-His-Tagged Proteins Using Novel Heterofunctional Chelate-Epoxy Supports. Biotechnology and Bioengineering, 2001. 76: p. 269.  R.D. Johnson and F.H. Arnold, Multiponit binding and heterogeneity in immobilized metal affinity chromatography. Biotechnology and Bioengineering, 1995. 48: p. 437.  C. Mateo, G. Fernandez-Lorente, B.C.C. Pessela, A. Vian, A.V. Carrascosa, J.L. Garcia, R. Fernandez-Lafuente and J.M. Guisan, Affinity chromatography of polyhistidine tagged enzymes new dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions. Journal of Chromatography A, 2001. 915: p. 97.  Y.-C. Liu, C.-C. ChangChien and S.-Y. Suen, Purification of penicillin G acylase using immobilized metal affinity membranes. Journal of Chromatography B, 2003. 794: p. 67.  Y.-C. Liu, S.-Y. Suen, C.-W. Huang and C.-C. Changchien, Effects of spacer arm on penicillin G acylase purification using immobilized metal affinity membranes. Journal of Membrane Science, 2005. 251: p. 201.  黃振翁, 應用多功能基薄膜純化及固定化盤尼西林醯胺酵素. 國立中興大學化學工程研究所碩士論文, 2004.  A. Kotha, L. Selvaraj, C.R. Rajan, S. Ponrathnam, K.K. Kumar, G.R. Ambekar and J.G. Shewale, Adsorption and expression of penicillin G acylase immobilized onto methacrylate polymers generated with varying pore generating solvent volume. Applied Biochemistry and Biotechnology, 1991. 30: p. 297.  A. Naidja, P.M. Huang and J.-M. Bollag, Activity of tyrosinase immobilized on hydroxyaluminum-montmorillonite complexes. Journal of Molecular Catalysis A:Chemical 1997. 115: p. 305.  J. He, X. Li, D.G. Evans, X. Duan and C. Li, A new support for the immobilization of penicillin acylase. Journal of Molecular Catalysis B: Enzymatic, 2000. 11: p. 45.  田蔚城, 生物技術的發展與應用. 九州圖書文物有限公司, 1998.  陳國誠, 生物固定化技術與產業應用. 茂昌圖書有限公司, 2000.  S. Hjerten, K. Yao, K.O. Erikson and B. Johasson, Gradient and isocratic high-perforemance hydrophobic interaction chromatography of proteins on agarose columns. Journal of Chromatography, 1986. 359: p. 99.  V.V. Mozhaev, Mechanism-based strategies for protein thermostabilization. Trends in Biotechnology, 1993. 11: p. 88.  R.M. Blanco and J.M. Guisan, Stabilization of enzymes by multipoint covalent attachment to agarose aldehyde gels-borohydride reduction of trypsin agarose derivatives. Enzyme and Microbial Technology, 1989. 11: p. 360.  E. Katchalski-Katzir, Immobilized enzymes: learning from past successes and failures. Trends in Biotechnology, 1993. 11: p. 471.  A. Schmid, J.S. Dordick, B. Hauer, A. Kiener, M. Wubbols and B. Wiyholt, Industrial biocatalysis today and tomorrow. Nature, 2001. 409: p. 258.  C. Mateo, O. Abian, R. Fern´andez-Lafuente and J.M. Guis´an, Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support–polyethyenimine composites. Biotechnology and Bioengineering 2000. 68: p. 98.  J. Turkova, Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function. Journal of Chromatography. B, Biomedical Sciences and Applications 1999. 1-2: p. 25.  J. Maly, C. Di Meo, M. De Francesco, A. Masci, J. Masojidek, M. Sugiura, A. Volpe and R. Pilloton, Reversible immobilization of engineered molecules by Ni-NTA chelators. Bioelectrochemistry, 2004. 63: p. 271.  C. Mateo, B. Pessela, M. Fuentes, R. Munilla and R. Fern´andez-Lafuente, Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports. Journal of Biotechnology, 2005. 120: p. 396.  C. Mateo, M. Fuentes, R. Munilla, A. V. Carrascosa and R. Fernandez-Lafuente, Purification and very strong reversible immobilization of large proteins on anionic exchangers by controlling the support and the immobilization conditions. Enzyme and Microbial Technology, 2006. 39: p. 909.  M. Yakup Arıca and 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: p. 255.  C. Mateo, O. Abian, R. Fernandez-Lafuente and J. M. Guisan, Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment. Enzyme and Microbial Technology, 2000. 26: p. 509.  C.-H. Chen, C.-W. Chen, C.-W. Huang and Y.-C. Liu, Simultaneous Purification and Immobilization of Penicillin G Acylase Using Bifunctional Membrane. Journal of Membrane Science, 2007: p. in press.  M.M. Bradford, 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.  張簡志強, 應用金屬親和薄膜分離純化盤尼西林醯胺酵素. 國立中興大學化學工程研究所碩士論文, 2003.  R.D. Johnson, R.J. Todd and F.H. Arnold, Multipoint binding in metal-affinity chromatography II. effect of pH and imidazole on chromatographic retention of engineered histidine-containing cytochromes c. Journal of Chromatography A 1996. 725: p. 225.  T. Cheng, M. Chen, H. Zheng, J. Wang, S. Yang and W. Jiang, Expression purification of penicillin G acylase enzyme from four different micro-organisms, and a comparative evaluation of their synthesis/hydrolysis ratios cephalexin. Protein Expression and Purification, 2006. 46: p. 107.  C. Mateo, O. Abian, R. Fernandez-Lafuente and 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: p. 739.  V. Grazu´, C. Mateo, F. Batista-Viera and R. Ferna´ndez-Lafuente, Stabilization of Enzymes by Multipoint Immobilization of Thiolated Proteins on New Epoxy-Thiol Supports. Biotechnology and Bioengineering, 2005. 90: p. 597.  W. Schwartza, M. Wysockib, L. Guerrierc and E.B. E. Birck-Wilson, Comparison of hydrophobic charge induction chromatography with affinity chromatography on protein A for harvest and purification of antibodies. Journal of Chromatography A, 2001. 908: p. 251.||摘要:||
本研究以再生纖維素薄膜（regenerated cellulose-based membrane）為固體載體，先使用EPI（epichlorohydrin）活化再生纖維素薄膜，使其表面帶有高反應性之環氧基，再利用化學合成的方式接上IDA（iminodiacetic acid）螯合劑，即為固定化金屬親和薄膜（immobilized metal affinity membrane, IMAM），並利用所得之金屬親和薄膜純化醱酵液中之盤尼西林醯胺酵素（Penicillin acylase, 簡稱PGA）。
第一部分探討再生纖維素薄膜表面改質之最適化條件及純化PGA之效果；在使用EPI活化薄膜過程中，NaOH濃度提高有利反應進行，但NaOH濃度在高於1.6M後，易造成再生纖維素薄膜變質，故EPI活化薄膜最適條件為20ml 1.4M NaOH, 5ml EPI, 24℃, 150rpm, 14h；而在使用IDA螯合劑之最適條件為25ml 1M IDA, 1M Na2CO3, pH 11, 24℃, 120rpm, 12h。以此條件改質之薄膜，銅離子螯合量為75μmol/disc、吸附PGA為1.8 IU/disc。所得之固定化酵素膜不需經長時間培育步驟，即可達到配位鍵結固定化酵素，此薄膜可在40天內，37℃重覆使用16次，不使用時保存含0.1﹪NaN3之PB（potassium phosphate buffer, pH 8）中，其PGA殘存活性可維持99﹪以上。而將10片固定化酵素膜置於流動式反應器純化PGA， PGA之回收率則可達97.1%，純化倍率為21.31倍。
在固定化酵素膜再生清洗步驟為，先使用Strip buffer（300mM NaCl, 100mM EDTA, 20mM Sodium phosphate buffer, pH8）浸泡30min去除膜上銅離子，接著0.5M HCl、0.5M NaOH分別浸泡10min去除膜上化合物、疏水性和親脂性蛋白質，經再生5次後，膜上PGA酵素活性殘存率仍有99﹪以上。
The regenerated cellulose-based membrane (RC membrane) was employed to construct the immobilized metal affinity membrane (IMAM) for purification and immobilization of penicillin G acylase (PGA).
This research is constituted of two parts. The first part was the preparation of IMAM on the RC membrane and the purifications of the PGA by using IMAM. For the preparation of IMAM, factors such as chelator density on the surface, epoxy functional group, NaOH concentration and elution solutions were investigated.
The epichlorohydrin (EPI) reaction was enhanced as increasing NaOH concentration. However, the RC membrane was deteriorated when the NaOH concertation was higher than 1.6M. The optimal reaction conditions were concluded as follows: for one RC membrane, 5ml EPI and 20ml 1.4M NaOH was used and the reaction conditions were 24℃, 150rpm for 14h. After that, the membrane was immersed in 25ml of 1M IDA (dissolved in 1M sodium carbonate) and allowed to react at 24℃ for 12h. The amount of Cu2+ chelated on IMAM under this condition was 75μmol/disc and the adsorbed PGA was 1.8 IU/disc. By applying this conditions to the flow experiments in a cartridge, a 21.31-times of purification in specific activity with 97.1﹪recovery for PGA purification was obtained. Meanwhile, the activity of immobilized PGA could be retained for more than 40 days under 16-times repeated use at 37℃.
The second part was to discuss the regeneration procedures of immobilized enzyme membrane. The regeneration was conducted by immersing the immobilized enzyme membrane in the strip buffer (300mM NaCl, 100mM EDTA, 20mM Sodium phosphate buffer, pH8)for 30 min to elute Cu2+ ion on the membrane. Then the membrane was immersed in 0.5M HCl and 0.5M NaOH respectively for 10 min to wash out the residual reactants, hydrophobic protein, etc. If was found that even under regeneration 5 times, the activity of the immobilized PGA membrane still could be maintained above 99%. This means a repeated use of the PGA immobilized on the membrane is feasible with a marked stability on the immobilized PGA activity.
|Appears in Collections:||化學工程學系所|
Show full item record
TAIR Related Article
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.