Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3571
DC FieldValueLanguage
dc.contributor孫幸宜zh_TW
dc.contributor楊芳鏘zh_TW
dc.contributor.advisor邱信程zh_TW
dc.contributor.author陳盈君zh_TW
dc.contributor.authorChen, Ying-Jiunen_US
dc.contributor.other中興大學zh_TW
dc.date2007zh_TW
dc.date.accessioned2014-06-06T05:32:10Z-
dc.date.available2014-06-06T05:32:10Z-
dc.identifierU0005-1707200616315300zh_TW
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dc.identifier.urihttp://hdl.handle.net/11455/3571-
dc.description.abstract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。zh_TW
dc.description.abstractIn 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.en_US
dc.description.tableofcontents一、 緒論-------------------------------------------------01 二、 文獻回顧---------------------------------------------02 2-1 酵素的簡介--------------------------------------------02 2-2 脂肪酶------------------------------------------------05 2-2-1 脂肪酶介紹------------------------------------------05 2-2-2 脂肪酶之催化反應機制--------------------------------06 2-2-3 脂肪酶的界面活化作用--------------------------------06 2-2-4 脂肪酶之應用----------------------------------------07 2-2-5 Candida rugosa酶------------------------------------08 2-3 酵素固定化--------------------------------------------11 2-3-1 酵素固定化介紹--------------------------------------11 2-3-2 酵素固定化之特性------------------------------------11 2-4 固定化之基材------------------------------------------12 2-4-1 基材的介紹------------------------------------------12 2-4-2 蒙脫土簡介------------------------------------------13 2-4-3 高分子改質蒙脫土------------------------------------15 2-5 X光繞射原理-------------------------------------------16 2-6 穿透式電子顯微鏡--------------------------------------17 三、 實驗方法與步驟---------------------------------------19 3-1 實驗藥品與儀器----------------------------------------19 3-1-1 實驗藥品--------------------------------------------19 3-1-2 實驗儀器--------------------------------------------20 3-2 蛋白質溶液之分析--------------------------------------20 3-2-1 測定POP-2000高分子是否會干擾蛋白質溶液分析----------20 3-2-2 以UV/Vis分光光譜儀測定蛋白質濃度之標準曲線----------20 3-2-3 不同pH值下,蛋白質溶液之表面電位測定----------------21 3-3 蛋白質固定於蒙脫土之分析測定--------------------------21 3-3-1 不同蛋白質添加量固定於蒙脫土------------------------21 3-3-2 蛋白質吸附於蒙脫土後之清洗量測試--------------------21 3-3-3 不同pH值下,蛋白質固定於蒙脫土----------------------22 3-4 利用XRPD測定蒙脫土之層間距----------------------------23 3-4-1 測定Na+-MMT及MMT-POP2000層間距----------------------23 3-4-2 測定蛋白質固定於蒙脫土後之層間距--------------------23 3-5 TGA測定-----------------------------------------------23 3-5-1 Na+-MMT、MMT-POP2000及lipase之TGA測定---------------23 3-5-2 Lipase在蒙脫土上固定量之TGA測定---------------------23 3-6 製備TEM的切片樣品-------------------------------------24 3-7 Lipase固定於蒙脫土前後,酵素的活性測定----------------24 3-7-1 製備測定酵素活性所需之溶劑--------------------------24 3-7-2 製備酵素和基質溶液----------------------------------24 3-7-3 測定基質溶液之標準曲線------------------------------25 3-7-4 不同lipase起始添加量對吸附於蒙脫土後之活性測定------25 3-7-5 酵素動力學研究--------------------------------------25 3-7-6 Lipase吸附於蒙脫土前後,酵素活性之最適溫度----------26 3-7-7 Lipase吸附於蒙脫土前後,酵素活性對溫度之穩定性測試--26 3-7-8 Lipase吸附於蒙脫土前後,酵素活性的貯存能力測試------27 四、 結果與討論-------------------------------------------28 4-1 蒙脫土基本性質----------------------------------------28 4-1-1 POP-2000對lipase溶液干擾之測定----------------------28 4-1-2 TGA測定---------------------------------------------30 4-1-3 XRPD測定--------------------------------------------31 4-1-4 蛋白質溶液之表面電位(Zeta-potential)測定------------32 4-2 Lipase於蒙脫土上之固定量------------------------------33 4-2-1 不同lipase添加量吸附於蒙脫土分析--------------------33 4-2-2 利用TGA計算lipase於蒙脫土上之固定量-----------------46 4-2-3 TEM分析---------------------------------------------50 4-2-4 Lipase吸附於蒙脫土後之清洗量測試--------------------52 4-3 pH值對不同添加量之lipase影響--------------------------56 4-4 Lipase吸附蒙脫土前後,酵素對基質之活性測定------------59 4-4-1 Lipase不同的起始添加量吸附於蒙脫土後對活性的影響----59 4-4-2 酵素動力學------------------------------------------61 4-5 溫度對酵素活性之影響----------------------------------63 4-5-1 Lipase吸附於蒙脫土前後,酵素活性之最適溫度----------63 4-5-2 Lipase吸附於蒙脫土前後,酵素活性對溫度之穩定性------65 4-5-3 Lipase吸附於蒙脫土前後,酵素活性的貯存能力----------67 五、 結論-------------------------------------------------70 參考文獻--------------------------------------------------71zh_TW
dc.language.isoen_USzh_TW
dc.publisher化學工程學系所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1707200616315300en_US
dc.subjectCandida rugosa lipaseen_US
dc.subject脂肪酶黏土zh_TW
dc.subjectclayen_US
dc.titleLipase與POP改質蒙脫土之交互作用及其酵素活性探討zh_TW
dc.titleInteractions of Lipase with ω-Diamino poly(oxypropylene) Intercalated Montmorillonites and its Effects on Enzymatic Activityen_US
dc.typeThesis and Dissertationzh_TW
item.languageiso639-1en_US-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.grantfulltextnone-
item.fulltextno fulltext-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
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