Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/99511
標題: 以天絲纖維不織布固定化金屬親和吸附基材進行純化及固定化過程
Tencel nonwoven fabric-based immobilized metal affinity chromatography adsorbents for enzyme purification and immobilization
作者: 曾韋寧
Wei-Ning Tseng
關鍵字: 天絲纖維;不織布;海藻糖合成酶;蛋白質純化;固定化金屬親和層析法;酵素固定化;Tencel;nonwoven fabric;trehalose synthase;protein purification;immobilized metal affinity chromatography;enzyme immobilization
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摘要: 
本研究以天絲纖維不織布做為固定化金屬親和吸附材之固定載體,接著以化學合成法依序接上環氧氯丙烷及螯合劑亞胺二乙酸(IDA),最後再接上過渡金屬離子,即完成固定化金屬吸附基材。
此金屬螯合親和吸附材之過渡金屬離子可以與含有poly His-tag 基因重組海藻糖合成酶的poly His-tag形成配位共價鍵,以達到純化及固定化海藻糖合成酶的目的。研究結果指出最適化的基材活化條件是在 6.2N 環氧氯丙烷與 3N 的氫氧化鈉於35°C持續反應六小時,得到的銅離子鍵結量為6771 μmol Cu(II)/ g-support;比較由六種不同金屬離子固定化後的海藻糖合成酶,以Zn (II)-loaded adsorbent呈現出最高的蛋白質吸附量為23.9 mg protein/g adsorbent與比活性為 15.9 U/ mg protein;粗酵素液含有300 mM NaCl具有最佳的選擇性,並且發現在吸附時的粗酵素液含有50 mM imidazole的純化效果比未添加時的純化效果更佳:以四種不同比例的Tencel/PET吸附海藻糖合成酶,發現添加PET會減少蛋白質吸附量但是可能會增加與蛋白質之間的吸附力。固定化海藻糖合成酶經過重複批次的使用,會因為酵素脫落問題導致活性下降。

In this study, Tencel nonwoven fabric-based immobilized metal affinity chromatography (IMAC) adsorbents were prepared upon activation with epichlorohydrin and conjugation with iminodiacetic acid. The results showed that the optimum surface activation was achieved with 6.2 N epichlorohydrin, 3 N sodium hydroxide at 35 ° C for six hours, giving a metal chealting capacity of 6771 μmol Cu(II)/ g-support. Among the six metal ions tested, the Zn (II) –loaded IMAC adsorbent exhibited the highest absorptive capacity and superior selectivity for the model protein, the poly(His)-tagged trehalose synthase, 23.9 mg protein/g adsorbent with a specific activity of 15.9 U/ mg protein. The Optimal selectivity was observed with 300 mM NaCl in the adsorption buffer. Furthermore, it was found that the inclusion of 50 mM imidazole in the adsorption buffer or wash buffer could significantly increase the purity of trehalose synthase. It was found that PET can reduced the adsorption capacity of the nonwoven fabric-based adsorbents but might enhance adsorption. Based on the above results, we can used for purification and immobilization of poly(His)-tagged proteins. A residual activity of approximately 71% was maintained after 20 cycles in a repeated-batch operation. The decline in residual activity was primarily due to the desorption of the trehalose synthase.
URI: http://hdl.handle.net/11455/99511
Rights: 同意授權瀏覽/列印電子全文服務,2019-08-29起公開。
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