Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3571
標題: Lipase與POP改質蒙脫土之交互作用及其酵素活性探討
Interactions of Lipase with ω-Diamino poly(oxypropylene) Intercalated Montmorillonites and its Effects on Enzymatic Activity
作者: 陳盈君
Chen, Ying-Jiun
關鍵字: Candida rugosa lipase
脂肪酶黏土
clay
出版社: 化學工程學系所
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摘要: 於本研究中,將脂肪酶(Candida rugosa lipase)固定於天然蒙脫土(Na+-MMT),及經高分子改質之蒙脫土(MMT-POP2000)上。由實驗結果發現在低lipase添加量(5~30 mg)時,lipase是以單層吸附方式固定於蒙脫土,在高lipase添加量(30~120 mg)時,是以多層吸附方式固定於蒙脫土。於XRPD實驗中,lipase固定於Na+-MMT上之層間距是隨添加量增加而上升,而lipase固定於MMT-POP2000上會因POP-2000被置換使得層間距先下降,再隨添加量增加而上升。由TEM分析可得知,無論是單層或多層吸附時,規則的層狀結構無改變。將固定於蒙脫土之lipase經由pH 9不同離子強度的緩衝溶液清洗後,可得知lipase單層吸附於Na+-MMT是以靜電作用力固定,lipase單層吸附於MMT-POP2000是疏水作用力與靜電作用力同時存在;而lipase多層吸附於蒙脫土時多以蛋白質間之疏水作用力固定。在不同pH值的環境下,lipase之吸附固定量會隨pH值增加而下降,Na+-MMT受pH值影響的比例比MMT-POP2000大。 在活性部份,單層吸附方式固定於Na+-MMT之酵素活性最差,而多層吸附方式固定於MMT-POP2000之酵素活性佳。根據Michaelis-Menten動力公式計算得知,純lipase之KM最小為0.0002 M,Kcat最大為0.1580 s-1;吸附固定後KM值會上升而Kcat值下降,代表吸附後基質與蛋白質接觸機會減少,且活性能力下降,而不同的吸附情形會有不同的下降程度。固定後lipase之最適溫度皆比未固定之lipase高,而單層吸附的保護效果較多層吸附為佳。固定後之lipase熱穩定性及貯存能力皆有明顯的提升。
In this study, protein, candida rugosa lipase, was adsorbed on sodium montmorillonite (Na+-MMT) and polyoxypropylene (POP) intercalated MMT (MMT-POP2000). The results show two different behaviors in adsprption isotherms, namely, the mono- (5~30 mg) and multi-layer (30~120 mg) types. From the XRPD measurement, the d-spacing of MMT increased slightly with the adsorption, but for MMT-POP2000, the expanded d-spacing collapsed during the adsorption process due to the libration of POP-2000 in the gallery of MMT. From TEM image, exfoliation did not occure by the adsorption of lipase on Na+-MMT and MMT-POP2000. By washing processes with buffer solution pH 9, it was found that lipase was immobilized on Na+-MMT mainly by electrostatic interaction, while the adsorption was driven by electrostatic and hydrophobic interaction on MMT-POP2000. However, hydrophobic association became more predominant in multilayer adsorption on MMT-POP2000. The lipase adsorption decreased with increasing of the pH of the adsorption media, and the influence of pH was more pronounced on Na+-MMT than MMT-POP2000. The enzymatic activity of lipase adsorbed on Na+-MMT in monolayer type was reduced to the largest extent while the lipase adsorbed in multilayers of MMT-POP2000 retained significant activity. The increase in KM is attributed to the increased steric hindrance against the interaction between the enzyme and the substrate while the decrease in Kcat indicates the partial conformational change of the adsorbed enzyme. The optimum temperature for the lipase activity was elevated after the adsorption. It was found that the lipases adsorbed in monolayer exhibited increased resistance in activity to the temperature increase. The lipase activity after the enzyme adsorption on Na+-MMT and MMT-POP2000 retained to a higher extent than the free counterpart in thermal stability and storage test.
URI: http://hdl.handle.net/11455/3571
其他識別: U0005-1707200616315300
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1707200616315300
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