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標題: 以矽膠固定化金屬親和吸附劑整合純化及固定化過程
Integrated enzyme purification and immobilization processes with silica-based immobilized metal affinity adsorbents
作者: Chen-Xin You
關鍵字: Protein purification;Enzyme immobilization;IMAC;蛋白質純化;酵素固定化;金屬親和層析
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這次的研究,是採用以矽膠膠體作為基材改質成雙官能基,同時具有能以 IMAC 吸附酵素末端含有 His-tag 的蛋白,以及能夠和酵素產生共價鍵鍵結的 epoxy 官能基;利用此方法,省去大量的時間及金錢成本,整合酵素純化及酵素固定化的步驟。
在此次研究結果中,得到的最適化的膠體比例為 1 : 5 之膠體,也就是銅離子的鍵結量為 88.42 μmol/g 的情況下,比起其他比例之膠體的吸附效果更好;且發現在吸附時的粗酵素液中含有 25 mM imidazole 時的純化效果比未添加時的純化效果更佳,同時也具有最佳的選擇率。
完成了以共價鍵固定之固定化酵素後進行反應 pH 、溫度及重複批次操作探討,最適化的操作條件在 pH 值為 8 ,反應溫度為 40°C ,重複操作的效果在經過 12 次的操作下,其相對活性也都落在 90 % 左右未見其顯著下降。

Silica-based immobilized metal affinity chromatography adsorbents with various ligand densities were prepared for the purification and immobilization of poly(His)-tagged proteins. An adsorbent with a ligand density of 88.42 μmol Cu2+/g gel exhibiting the optimal selectivity. And the crude enzyme was added 25 mM imidazole the purification better results than when not added, but also has the best selectivity.
After the completion of immobilization enzyme, investigate the reaction pH, temperature and reusability. The optimal reaction temperature and reaction pH of the immobilized enzyme were identified as 40°C and 8.0. The effect of reusability after 12 operations, the relative activity is about 90% and no significant decline.
Based on the above results, we successfully establishing that this system can be used for the direct purification and immobilization of poly(His)-tagged proteins.
其他識別: U0005-1308201517244600
Rights: 同意授權瀏覽/列印電子全文服務,2018-08-19起公開。
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