臺灣博碩士論文加值系統

本研究使用泛素和silaffin R5的融合蛋白(U-R5)為催化劑，並於磁性奈米粒子存在下，以經酸水解的四甲氧基甲矽烷(TMOS)作為矽酸來源，生成矽氧以進行Candida rugosa脂解酶的固定化。為使固定達最適化，分別探討不同U-R5、奈米磁性粒子(MNP)、脂解酶、和經酸水解的TMOS添加量對包覆率和固定後脂解酶之比活性的影響。
當U-R5、脂解酶和經酸水解TMOS的添加量增加時，包覆率會隨之遞減，但固定後脂解酶之比活性卻反而提升。反之，添加MNP的濃度由1 mg/ml增加至4 mg/ml時，包覆率和比活性皆會隨之提升，不過增至5 mg/ml時，後者反而下降。使用7.5 μM U-R5和30 μM聚烯丙胺(PAA)所導引的脂解酶固定化具有相似的包覆率(~60%)，脂解酶比活性於固定前後亦相似。然而當兩者添加量增加時，卻產生相反效果。較高的PAA添加量使包覆率較佳，然而比活性卻較低。

In this study, immobilization of Candida rugosa lipase was performed using ubiquitin and silaffin R5 fusion protein, U-R5, as a catalyst and hydrolyzed tetramethoxysilane (TMOS) as the source of silicic acid to generate silica in the presence of magnetic nanoparticle (MNP).To optimize the immobilization, the effects of the amount of U-R5, MNP, lipase and hydrolyzed TMOS added on the encapsulation efficiency and the specific activity of lipase after immobilization were investigated.
When U-R5, lipase and hydrolyzed TMOS added were increased, the encapsulation efficiencies were decreased but the specific activities of immobilized lipase were increased. Conversely, as the concentrations of MNP added were increased from 1 up to 4 mg/ml, the encapsulation efficiencies and the specific activities both were increased. However, the latter becamelowerat the MNP concentration of 5 mg/ml. The use of 7.5 μM U-R5 or 30 μM PAA to mediate immobilization of lipase gave similar encapsulation efficiencies (~60%), and the specific activitiesof immobilized lipase obtained were also similar to those of free lipase. Nevertheless, the increase in the addition amount of U-R5 and PAA resulted in the contrary effect; Higher PAA concentration displayed better encapsulation efficiency but lower specific activity.

誌謝ⅰ
摘要ⅱ
ABSTRACTⅲ
目次ⅳ
表次ⅵ
圖次ⅶ
第一章 緒論1
1.1 前言1
1.2 研究目的2
第二章 文獻回顧4
2.1 酵素4
2.2 脂解酶5
2.3 泛素7
2.4 酵素固定化8
2.4.1 酵素固定化之簡介8
2.4.2 固定化方法9
2.5 仿生矽化11
第三章 材料與方法14
3.1 實驗藥品14
3.2 實驗設備15
3.3 實驗方法16
3.3.1 U-R5融合蛋白的表現16
3.3.2 U-R5蛋白純化 16
3.3.3蛋白質定量19
3.3.4 SDS-PAGE分析 20
3.3.5脂解酶的活性分析方法24
3.3.6以仿生矽化固定酵素25
3.3.6.1製備磁性奈米粒子25
3.3.6.2脂解酶的固定化26
3.3.6.3二氧化矽的定量27
第四章 結果與討論29
4.1 U-R5的生產表現 29
4.2固定條件的最適化29
第五章 結論32
第六章 圖表33
第七章 參考文獻56

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