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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
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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。

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.
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