Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3680
標題: 酵素與POP改質蒙脫土之交互作用及酵素活性探討
Interactions of enzymes with α,ω-diaminopoly(oxypropylene) intercalated montmorillonite and their effects on the enzymatic activity
作者: 陳國井
Chen, Guo-Jing
關鍵字: protein;蛋白質;enzyme;immobilzation;montmorillonite;activity;酵素;固定;蒙脫土;活性
出版社: 化學工程學系所
引用: 參考文獻 (1) Park, J. B. 1984. Biomaterials Science and Engineering. New York, NY: Plenum Press, Chapters 1 and 7. (2) Duncan, E. 1990. “Biomaterials. What is a Biomaterial?” Med. Dev. Diag. Ind., 12, 138-142. (3) Brook, S. D. 1980. Properties of Biomaterials in Physiological Environment. Boca Ration, FL: CRC Press, Chapter 4. (4) Williams, D. F. 1991. The Significance of Surface in Biocompatibility Phenomena. Surfaces in Biomaterials Symposium, 1-4. (5) Anderson, J. M. 1986. In vivo Biocompatibility Studies: Perspectives on the Evaluation of Biomedical Polymer Biocompatibility. Polymeric Biomaterials, 29-39. (6) Anderson, J. M. 1988. Inflammatory response to Implants. Trans Am. Soc. Artif. Intern. Organs 34, 101-107. (7) Olphen, H.V., (1977) Clay Colloid Chemistry, Chapter V, Wiley-Interface publication, New york. (8) Konta, J. (1995) Clay and man: Clay raw materials in the service of man. Appl. Clay Sci. 10, 275-335. (9) Bolger, R. (1995) Industrial minerals in pharmaceuticals, Ind. Min., 52-63. (10) Carretero, M.I. (2002) Clay minerals and their beneficial effects upon human health. A review, Appl. Clay Sci., 21,155-163. (11) Gamiz E., Linares J., Delgado R. (1992) Assessment of two Spanish bentonites for pharmaceutical uses, Appl. Clay Sci. 6, 359-368. (12) Lin, F.H., Lee, Y.H., Jian, C.H., Wong, J.M., Shieh, M.J., Wang, C.Y. (2002) A study of purified montmorillonite intercalated with 5-fluorouracil as drug carrier, Biomaterials 23,1981-1987. (13) Cara, S., Carcangiu, G., Padalino, G., Palomba, M., Tamanini, M. (2000) The bentonites in pelotherapy: thermal properties of clays pastes from Sardinia, Appl. Clay Sci., 16, 125-132. (14) Poensin, D., Carpentier, P.H., Fechoz, C., Gasparini, S. (2003) Effects of mud pack treatment on skin microcirculation, Jt. Bone Spine 70, 367-370. (15) Summa, V., Tateo, F. (1998) The use of pelitic raw materials in thermal centres: mineralogy, geochemistry, grain size and leaching test. Examples from the Lucania area, Appl. Clay Sci. 12, 403-417. (16) Castela-Papin, N., Cai, S., Vatier, J., Keller, F., Souleau, C.H., Farinotti, R. (1999) Drug interaction with diosmectite: a study using the artificial stomach–duodenum model, Int. J. Pharm. 182, 111-119. (17) del Hugo, C., Vincente, M.A., Rives, V. (2001) Preparation of drug-montmorillonite UV-radiation compounds by gas–solid adsorption, Clay Miner. 36, 541-546. (18) Ito, T., Sugafuji, T., Maruyama, M., Ohwac, Y., Takahashic, T. (2001) Skin penetration by indomethacin is enhanced by use of an indomethacin/smectite complex, J. Supramol. Chem. 1, 217-219. (19) McCarter, W.J., Broussean, R. (1990) The A.C. responses of hardened cement paste, Cem. Concr. Res. 20, 891-900. (20) White, J.L., Hem, S.L. (1983) Pharmaceutical aspect of clay–drug interactions, Ind. Eng. Chem. Prod. Res. Dev. 22, 665-671. (21) Lee, W.F., Fu, Y.T. (2003) Effect of montmorillonite on the swelling behavior and drug-release behavior of nanocomposite hydrogels, J. Appl. Polym. Sci. 89, 3652-3660. (22) Wilson, M.J. (1987) A Handbook of Determinative Methods in Clay Mineralogy, Chapman and Hall, New York, USA. (23) Elmore, A.R. (2003) Final report on the safety assessment of aluminum silicate, calcium silicate, magnesium aluminum silicate, magnesium silicate, magnesium trisilicate, sodium magnesium silicate, zirconium silicate, attapulgite, bentonite, Fuller''s earth, hectorite, kaolin, lithium magnesium silicate, lithium magnesium sodium silicate, montmorillonite, pyrophyllite, and zeolite, Int. J. Toxicol. 22, Suppl 1, 37-102. (24) Ray, S.S., Okamoto, M. (2003) Polymer/layered silicate nanocomposites: a review from preparation to processing, Prog. Polym. Sci. 28, 1539-1641. (25) Ferrario, J.B., Byrne, C.J., Cleverly, D.H. (2000) 2,3,7,8-dibenzo-p -dioxins in mined clay products from the United States: evidence for possible natural origin, Environ. Sci. Technol. 34, 4524-4532. (26) Abdel-Wahhab, M.A., Nada, S.A., Farag, I.M., Abbas, N.F., Amra, H.A. (1998) Potential protective effect of HSCAS and bentonite against dietary aflatoxicosis in rat: with special reference to chromosomal aberrations, Nat. Toxins 6, 211-218. (27) Huwig, A., Freimund, S., Kappeli, O., Dutler, H. (2001) Mycotoxin detoxication of animal feed by different adsorbents, Toxicol. Lett. 122, 179-188. (28) Armstrong, A., Aden, K., Amraoui, N., Diekkruger, B., Jarvis, N., Mouvet, C., Nicholls, P., Wittwer, C. (2000) Comparison of the performance of pesticide-leaching models on a cracking clay soil: results using the Brimstone Farm dataset, Agric. Water Manag. 44, 85-104. (29) Brown, C.D., Fryer, C.J., Walker, A. (2001) Influence of topsoil tilth and soil moisture status on losses of pesticide to drains from a heavy clay soil, Pest Manag. Sci. 57, 1127-1134. (30) Hocine, O., Boufatit, A., Khouider, A. (2004) Use of montmorillonite clays as adsorbents of hazardous pollutants, Desalination 167, 141-145. (31) Maxwell, D.J., Taylor, J.R., Nie, S.M. (2002) Self-assembled nanoparticle probes for recognition and detection of biomolecules, J. Am. Chem. Soc. 124, 9606-9612. (32) Sanchez-Martin, M.J., Rodriguez-Cruz, M.S., Andrades, M.S., Sanchez-Camazano, M. (2006) Efficiency of different clay minerals modified with a cationic surfactant in the adsorption of pesticides: influence of clay type and pesticide hydrophobicity, Appl. Clay Sci. 31, 216-228. (33) Lan, T., Kaviratna, P.D., Pinnavaia, T. (1995) Mechanism of clay tactoid exfoliation in epoxy-clay nanocomposites. Chem. Mater. 7, 2144-2150. (34) Messersmith, P.B., Giannelis, E.P. (1994) Synthesis and Characterization of Layered Silicate-Epoxy Nanocomposites Chem. Mater. 6, 1719-1725. (1994). (35) Yano, K., Usuki, A., Okada., A. (1997) Synthesis and properties of polyimide-clay hybrid films. J. Polym. Sci., Part A:Polym. Chem. 35, 2289-2294. (36) Tyan, H.L., Liu, Y.C., Wei. K.H. (1999) Enhancement of imidization of poly(amic acid) through forming poly(amic acid)/organoclay nanocomposites Polymer 40, 4877-4886. (37) Al-Esaimi, M.M. (1997) Reaction catalyzed by montmorillonite: Polymerization of methyl methacrylate. J. Appl. Polym. Sci. 64, 367-372. (38) Cai, P., Huang, Q., Zhang, X., Chen, H. (2006) Adsorption of DNA on clay minerals and various colloidal particles from an Alfisol. Soil Bio. Biochem. 38, 471-476. (39) Vettori, C., Paffetti, D., Pietramellara, G., Stotzky, G., Gallori, E. (1996) Amplification of bacterial DNA bound on clay minerals by the random amplified polymorphic DNA (RAPD) technique. FEMS Microbio. Ecol. 20, 251-260. (40) Cai, P., Huang, Q., Chen, W., Zhang, D., Wang, K., Jiang, D., Liang, W. (2007) Soil colloids-bound plasmid DNA: Effect on transformation of E. coli and resistance to DNase I degradation. Soil Biol. Biochem. 39, 1007-1013. (41) Lin, F.H., Chen, C.H., Cheng, Winston T.K., Kuo, T.F. (2006) Modified montmorillonite as vector for gene delivery. Biomaterials 27, 3333-3338. (42) Vettori, C., Calamai, L., Yoder, M., Stotzky, G., Gallori, E. (1999) Adsorption and binding of AmpliTaq® DNA polymerase on the clay minerals, montmorillonite and kaolinite. Soil Biol. Biochem. 31, 587-593. (43) Pietramellara, G., Dal Canto, L., Vettori, C., Gallori, E., Nannipieri, P. (1997) Effects of air-drying and wetting cycles on the transforming ability of DNA bound on clay minerals. Soil Bio. Biochem. 29, 55-61. (44) Liu, Y., Huang, J., Wang, X. (2006) Adsorption of chlorophyll and carotene on attapulgite, montmorillonite and activated carbons from soft oil. Kuangwu Yanshi 26, 12-15. (45) Kakegawa, N., Ogawa, M. (2002) The intercalation of β-carotene into the organophilic interlayer space of dialkyldimethylammonium- montmorillonites. Applied Clay Science 22, 137-144. (46) Sarier, N., Guler, C. (1989) The mechanism of β-carotene adsorption on activated montmorillonite. J. Am. Oil Chem. Soc. 66, 917-23. (47) Jian, C.H., Lin, F.H., Lee, Y.H. (2000) A study of purified montmorillonite intercalated with 5-fluorouracil as drug carrier. Biomedical Sciences Instrumentation 36, 391-396. (48) Lin, F.H., Lee, Y.H., Jian, C.H., Wong, J.M., Shieh, M.J., Wang, C.Y. (2002) A study of purified montmorillonite intercalated with 5-fluorouracil as drug carrier. Biomaterials 23, 1981-1987. (49) Yang, C., Pi, Z.B., Tian, X.K., Wang, C.J., Yang, L.J., Li, H., Lin, Y.P. (2002) Experiment study of ultra-fine montmorillonite used as a controlled-release carrier of fluorouracil. Wuhan Daxue Xuebao, Lixueban 48, 652-654. (50) Akalin, E., Akyuz, S., Akyuz, T. (2007) Adsorption and interaction of 5-fluorouracil with montmorillonite and saponite by FT-IR spectroscopy. Journal of Molecular Structure 834-836, 477-481. (51) Lozzi, I., Calamaia, L., Fusia, P., Bosettoa, M., and Stotzky, G. (2001) Interaction of horseradish peroxidase with montmorillonite homoionic to Na+ and Ca2+: effects on enzymatic activity and microbial degradation. Soil Biology & Biochemistry 33, 1021-1028. (52) Naidja, A., Huang, P.M., Bollag, J.-M. (1997) Activity of tyrosinase immobilized on hydroxyaluminum-montmorillonite complex. J. Mol. Catal. A: Chemical 115, 305-316. (53) Naidja, A., Huang, P.M. (1996) Deamination od aspartic acid by aspartase-Ca-montmorillonite complex. J. Mol. Catal. A: Chemical 106, 225-265. (54) Usuki, A., Kawasumi, M., Kojima, Y., Okada, A., Kurauchi, T. (1993) Swelling behavior of montmorillonite cation exchange for ω-amino acid-b-ε-caprolactam. J. Mater. Res. 8, 1174-1187. (55) Lan, T., Kaviratna, P. D., Pinnavaia, T. J. (1996) Epoxy self-polymerization in smectite clays. J. Phys. Chem. Solids 57, 1005-1010. (56) Wang, M. S., Pinnavia, T. J. (1994) Clay-polymer nanocomposites formed from acidic derivatives of montmorillonite and an epoxy resin. Am. Chem. Soc. 6, 468-473. (57) Wang, Z., Pinnavia, T. J. (1998) Hybrid Organic-inprganic nanocomposites: exfoliation of magadiite nanolayer in elastomeric epoxy polymer. Chem. Mater. 10, 1820-1826. (58) Chen, T. K., Tien, Y. I., Wei, K. H. (2000) Synthesis and characterization of novel segmented polyurethane/clay nanocomposite. Polymer 41, 1345-1353. (59) Lin, J.-J., Cheng, I.-J., Wang, R., and Lee, R.-J. (2001) Tailoring basal spacings of montmorillonite by poly(oxyalkylene)diamine intercalation. Macromolecules 34, 8832-8834. (60) Chou, C.-C., Shieu, F.-S., and Lin, J.-J. (2003) Preparation, organophilicity, and self-assembly of poly(oxypropylene)amine-clay hybrids. Macromolecules 36, 2187-2189. (61) Lin J.-J., Chen, I.-J., and Chou, C.-C. (2003) Critical conformational change of poly(oxypropylene)diamines in layered aluminosilicate confinement. Macromol. Rapid Commun. 24, 492-495. (62) Choy, J.-H., Kwak, S.-Y., Jeong, Y.-J., and Park J.-S. (2000) Inorganic layered double hydroxides as nonviral vectors. Angew. Chem., Int. Ed. 39, 4041-4045. (63) Desigaux, L., Belkacem, M. B., Richard, P., Cellier, J., Léone, P., Cario, L. Leroux, F. Taviot-Gueho, C., and Pitard, B. (2006) Self-assembly and characterization of layered double hydroxide/DNA hybrids. Nano Lett. 6, 199-204. (64) Kumar, C. V., and Chaudhari, A. (2000) Protein immobilized at the galleries of layered α-zirconium phosphate: Structure and activity studies. J. Am. Chem. Soc. 122, 830-837. (65) Kumar, C. V., and Chaudhari, A. (2002) High temperature peroxidase activities of HRP and hemoglobin in the galleries of layered Zr(IV) phosphate. Chem. Commun. 20, 2382-2383. (66) Kumar, C. V., and Chaudhari, A. (2003) Unusual thermal stabilities of some proteins and enzymes bound in the galleries of layered α-Zr(IV)phosphate/phosphonates. Microporous Mesoporous Mater. 57, 181-190. (67) Bellezza, F., Cipiciani, A. Costantino, U., and Negozio, M. E. (2002) Zirconium phosphate and modified zirconium phosphates as supports of Lipase. Preparaption of the composites and activity of the supported enzyme. Langmuir 18, 8737-8742. (68) Bellezza, F., Cipiciani, A., Costantino, U., Nicolis, S. (2004) Catalytic activity of myoglobin immobilized on zirconium phosphonates. Langmuir 20, 5019-5025. (69) Bellezza, F., Cipiciani, A., Costantino, U., Marmottini, F. (2006) Adsorption of myoglobin onto porous zirconium phosphate and zirconium benzenephosphonate obtained with template synthesis. Langmuir 22, 5064-5069. (70) Kumar, C. V., and Chaudhari, A. (2001) Efficient renaturation of immobilized Met-hemoglobin at the galleries of α-zirconium phosphate. Chem. Mater. 13, 238-240. (71) Baron, M. H., Revault, M., Servagent-Noinville, S., Abadie, J., and Quiquampoix, H. (1999) Chymotrypsin adsorption on montmorillonite: Enzymatic activity and kinetic FTIR structure analysis. J. Colloid Interface Sci. 214, 319-332. (72) Ding, X., and Henrichs, S. M. (2002) Adsorption and desorption of proteins and polyamino acids by clay minerals and marine sediments. Mar. Chem. 77, 225-237. (73) Chiu, H.-C., Zalipsky, S., Kopečková, P., and Kopeček, J. (1993) Enzymatic activity of chymotrypsin and its poly(ethylene glycol) conjugates toward low and high molecular weight substrates. Bioconjugate Chem. 4, 290-295. (74) Mattews, B. W., Sigler, P. B., Henderson, R., and Blow, D. M. (1967) Three-dimensional structure of tosyl-α-chymotrypsin. Nature 214, 652-656. (75) Cristofaro, A. D., and Violante, A. (2001) Effect of hydroxy-aluminium species on the sorption and interlayering of albumin onto montmorillonite. Appl. Clay Sci. 19, 59-67. (76) Calamai, L., Lossi, I., Stotzky, G., Fusi, P., and Ristori, G. G. (2000) Interaction of catalase with montmorillonite homoionic to cations with different hydrophobicity: Effect on enzymatic activity and microbial utilization. Soil Biol. Biochem. 32, 815-823. (77) Fusi, P., Ristori, G. G., Calamai, L., and Stotzky, G. (1989) Adsorption and binding of protein on “clean” (homoionic) and “dirty” (coated with Fe oxyhydroxides) montmorillonite, illite and kaolinite. Soil Biol. Biochem. 21, 911-920. (78) Spek, E. J., Bui, A. H., Lu, M., and Kallenbach N. R. (1998) Surface salt bridges stabilize the GCN4 leucine zipper. Protein Sci. 7, 2431-2437. (79) Zhou, H.-X., and Dill, K. A. (2001) Stabilization of proteins in confined spaces. Biochemistry 40, 11289-11293. (80) Khechinashvili, N. (1990) Thermodynamic properties of globular proteins and the principle of stabilization of their native structure. Biochim. Biophys. Acta, Protein Struct. Molec. Enzym. 1040, 346-354. (81) Asthagiri, D., and Lenhoff, A. M., (1997) Influence of structure details in modeling electrostatically driven protein adsorption. Langmuir 13, 6761-6768. (82) Lin, J.-J., Wei, J.-C., Juang, T.-Y., and Tsai, W.-C. (2007) Preparation of protein-silicate hybrids from polyamine intercalation of layered montmorillonite. Langmuir 23, 1995-1999. (83) Mattson, F.H. and Volpenheim, R.A. (1969) Relative rate of hydrolysis by rat pancreatic lipase of ester of C2-C18 fatty acids with C1-C18 primary normal alcohols. J. Lipid Res. 10, 271-276. (84) Vitro, M.D., Agud, I., Montero, S., Blanco, A., Solozabal, R., Lascary, J.M., Llama, M.J., Serra, J.L., Landeta, L.C., de Renobales, M. (1994) Hydrolysis of animal fats by immobilized candida rugosa lipase. Enzyme Microb. Technol. 16, 61-65. (85) Undurraga, D., Markovits, A., Erazo, S. (2001) Cocoa butter equivalent through enzymatic interesterification of palm oil midfraction. Process Biochem. 36, 933–939. (86) Plou, J.F., Barandiaran, M., Calvo, M.V., Ballesteros, A., Pastor, E. (1996) High yield production of mono and dioleylglycerol by lipase catalyzed hydrolysis of triolein. Enzyme Microb. Technol. 18, 66–71. (87) Dandik, L. and Aksoy, H.A. (1996) Applications of Nigella sativa seed lipase in oleo-chemical reactions. Enzyme Microb. Technol. 19, 277–281. (88) Yan, Y., Bornscheuer, U.T., Cao, L., Schmid, R.D. (1999) Lipasecatalyzed solid-phase synthesis of sugar fatty acid esters: removal of byproducts by azeotropic distillation. Enzyme Microb. Technol. 25, 725–728. (89) Watanabe, Y., Miyawaki, Y., Adachi, S., Nakanishi, K., Matsuno, R. (2001) Continuous production of acyl mannoses by immobilized lipase using a packed-bed reactor and their surfactant properties. Biochem. Eng. J. 8, 213–216. (90) Jaeger, K.E. and Reetz, T.M. (1998) Microbial lipases from versatile tools for biotechnology. Trends Biotechnol. 16, 396–403. (91) Maugard, T., and Legoy, M.D. (2000) Enzymatic synthesis of derivatives of Vitamin A in organic media. J. Mol. Catal. Part B. Enzym. 8, 275–280. (92) Grochulski, P., Li, Y., Schrag, J.D., Cygler, M. (1992) Two conformational states of Candida rugosa lipase. Protein Sci. 3, 82-91. (93) Brady, L., Brzozowski, A.M., Derewenda, Z.S., Dodson, E., Dodson, G., Tolley, S., Turkenburg, J.P., Christiansen, S., Huge-Jensen, B., Norskov, L., Thim, L., Menge, U. (1990) A serine protease triad forms the catalytic centre of a triacylglycerol lipase. Nature 343, 767-700. (94) Viseras, C., Meeten, G.H., Lo´pez-Galindo, A. (1999) Pharmaceutical grade phyllosilicate dispersions: the influence of shear history on floc structure. Int. J. Pharm. 182, 7-20. (95) de Fuentes, I.E., Viseras, C.A., Ubiali, D. Terreni, M., Alcántara, A.R. (2001) Different phyllosilicates as supports for lipase immobilization. J. Mol. Catalysis. B, Enzymatic 11, 657-663. (96) Montero, S., Blanco, A., Virto, M.D., Landeta, L.C., Agud, A., Solozabel, R., Lascaray, J.M., De Renobles, M., Llama, M.J., Serra, J.L. (1993) Immobilization of Candida rugosa lipase and some properties of the immobilized enzyme. Enzyme Microb. Technol. 15, 239-247. (97) Huang, F.-C., Ju, Y.-H. (1994) Improved activity of lipase by vacuum dryin on hydrophobic microporous support, Biotechnol. Tech. 8, 827-830. (98) Bailie, P.M., McNerlan, S.E., Robinson, E., Murphy, W.R. (1995) The immobilization of Candida rugosa lipase for the hydrolysis of fat and oils. IChemE. 73, 71-76. (99) Bosley, A. and Pielow, A. (1997) Immobilization of lipases on porous polypropylene: Reduction in esterification efficiency at low loading. J. Am. Oil. Chem. Soc. 74, 107-111. (100) Foresti, M.L. and Ferreira, M.L., 2007. Analysis of the interaction of lipase with polypropylene of different structure and polypropylene-modified glass surface. Colloids and Surface A: Physicochem. Eng. Aspects 294, 147-155. (101) Gougeon, R.D., Soulard, M., Reinholdt, M., Brendle, J.M-., Cheezeau, R., LeDred, R., Marchal, R., Jeandet, P. (2003) Polypeptide adsorption on a synthetic montmorillonite: a combined solid-state NMR spectroscope, X-ray diffraction, thermal analysis and N2 adsorption study. Eur. J. Inorg. Chem. 1366-1372. (102) Foresti, M.L. and Ferreira, M.L. (2005) Solvent-free ethyl oleate synthesis mediated by lipase from Candida Antarctica B adsorbed on polypropylene powder. Catalysis today 107-108, 23-30. (103) Diogo, M.M., Silva, S., Cabral, J.M.S., Quieroz, J.A. (1999) Hydrophobic interaction chromatography of Chromobacterium viscosum lipase on polypropylene glycol immobilised on Sepharose. Journal of Chromatography A 849, 413-419. (104) Palomo, J.M., Muňoz, G., Fernández-Lorente, G., Mateo, C., Fernández-Lorente, R., Guisan, J.M. (2002) Interfacial adsorption of lipases on very hydrophobic support (octadecyl-Sepabeds): immobilization, hyperactivation and stabilization of the open form of lipase, J. Mol. Catal. B: Enzym. 19/20, 279-286. (105) Wang, Z.G., Wang, J.Q., Xu, Z.K. (2006) Immobilization of lipase from Candida rugosa on electrospun polysulfone nanofibrous membranes by adsorption. J. Mol. Catal. B: Enzym. 42, 45-51. (106) Guo, Z., Bai, S., Sun, Y. (2003) Preparation and characterization of immobilized lipase on magnetic hydrophobic microspheres. Enzyme Microb. Technol. 32, 776-782.
摘要: 
中文摘要
天然蒙脫土經疏水性高分子α,ω-diaminopoly(oxypropylene) (POP)插層改質後,其層間距由原本之1.2 nm增大至5.5 nm。於本研究中,將利用此種改質後之雙性蒙脫土(POP/MMT)來固定蛋白質酵素,並探討其固定之情形與酵素活性變化,而於本研究中,蛋白質酵素主要分親水性蛋白質酵素及疏水性蛋白質酵素兩方面來探討。
由實驗結果中可知,利用天然蒙脫土來固定親水性蛋白質酵素chymotrypsin (CHT)時,蛋白質僅吸附於蒙脫土表面,而利用改質蒙脫土POP/MMT來固定蛋白質酵素時,蛋白質發生插層而進入層間中,而吸附量超過一臨界點(86μmol/g clay)後,蛋白質之吸附量及由X光粉末繞射儀所測得之層間距因親水性蛋白質酵素的完全插層而發生驟增的現象。由蛋白質酵素之活性結果中可以得知,完全插層於層間中的親水性蛋白質酵素對小分子量基質能保有完整的酵素活性,而與大分子量之蛋白質基質(BAS和catalase)反應時,具有極佳之保護效果。此外,此一插層後之蛋白質/蒙脫土奈米混合物具有良好之穩定效果,在使用後不會與其他生物分子發生置換或是發生脫層之現象。因此,隨著蛋白質酵素插層進入蒙脫土層間中,可以有效的將此一新型態之蛋白質/蒙脫土奈米混合物利用在藥物治療或是組織工程。
利用天然蒙脫土與改質蒙脫土來固定疏水性蛋白質酵素lipase時,疏水性蛋白質酵素出現兩階段之吸附現象。第一階段時,蛋白質酵素單層吸附於蒙脫土表面,而第二階段時,蛋白質酵素以多層吸附之方式固定。於單層吸附及多層吸附時,疏水高分子POP都有增加吸附固定量的功能。藉由高pH值緩衝溶液清洗之方式可知,疏水性蛋白質酵素是以靜電作用力固定於天然蒙脫土上,不同地,疏水性蛋白質以和疏水性高分子間之疏水作用力及蒙脫土間之靜電作用力而固定於改質蒙脫土POP/MMT上,於多層吸附時,則以蛋白質間之疏水作用力而進行固定。由活性結果中可知,利用POP/MMT來固定疏水性蛋白質酵素時,疏水高分子POP可以有效增加蛋白質酵素之活性,且於熱穩定性及儲存效果上也有明顯的改善,因此,於疏水性蛋白質酵素之應用上,改質蒙脫土POP/MMT也是一種良好之載體。

Abstrate
The basal spacing of sodium montmorillonite (Na+-MMT) was enlarged from 1.2 to 5.5 nm by a simple exchange of sodium ion in the gallery with α,ω-diaminopoly(oxypropylene) (POP). In this study, the modified MMT was used for enzyme immobilization, and the activity of the immobilized enzyme was also discussed.
For Na+-MMT, the protein molecules (α-chymotrypsin) were adsorbed on the external surfaces of the particles by the Langmuir-type adsorption. For POP/MMT, the effective intercalation of protein molecules within the galleries of montmorillonites could be achieved via simple exchange processes of POP. The basal spacing in the multilayered structure of clay was abruptly enlarged when the extent of protein intercalation increased to the critical point (86 μmol/g clay). The nanohybrids showed well preserved catalytic activity in hydrolyzing small substrates while establishing a barrier to interactions with large biomacromolecules. Furthermore, the structural stability of the inorganic/organic nanohybrids was enhanced so that either the exchange of biomolecules or the exfoliation of layered clay particles would not occur when exposed to other proteins (BSA and catalase). The results indicate that, through the benign accommodation of protein species between the inorganic platelets, this nanoscaled manipulation of protein functions could be highly useful in developing new inorganic/enzyme nanohybrids for protein therapeutics and tissue engineering.
The hydrophobic protein (lipase) was adsorbed on the surface of MMT particles by monolayer adsorption for the first step and by multilayer adsorption for the second step. The capacity of adsorption for MMT could be improved by the hydrophobic polymer POP. The lipase molecules were adsorbed on Na+-MMT particles by electrostatic interaction with MMT plates, and on POP/MMT hybrids by hydrophobic interaction with POP molecules. For multilayer adsorption, the lipase molecules were adsorbed on the surface by protein-protein interaction. From enzymatic activity results, the POP/MMT nanohybrids could improve advanced protection on the thermal and storage stability of the hydrophobic enzyme. From the results, it indicated that the nanohybrids could be used as the support for the immobilization of hydrophobic enzyme.
URI: http://hdl.handle.net/11455/3680
其他識別: U0005-1407200817210400
Appears in Collections:化學工程學系所

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