Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/91528
標題: 以絲瓜菜瓜布固定化金屬親和吸附基材進行蛋白質純化之研究
A luffa sponge-based immobilized metal ion affinity chromatography adsorbent for protein purification
作者: Wei-Jie Lai
賴葦潔
關鍵字: Luffa sponges;Hydroxyethyl cellulose;Immobilized metal ion affinity chromatography;Trehalose synthase;絲瓜菜瓜布;羥乙基纖維素;固定化金屬親和層析法;海藻糖合成酶
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摘要: 
本研究以絲瓜菜瓜布做為固定化金屬親和吸附材之固體載體,並利用羥乙基纖維素交聯於基材表面,以增加其表面氫氧基團,接著以化學合成法依續接上環氧氯丙烷、螯合劑亞胺二乙酸(IDA)以及過渡金屬離子,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之目的。由Langmuir model 可得知金屬螯合親和吸附材最大吸附量為2.45 mg/g及解離常數為0.195 mg/ml。比較由六種不同金屬離子固定化後的海藻糖合成酶,以Co (II)- loaded adsorbent 呈現最高的總活性(15.59 ± 0.74 U/g) 與比活性 (3.66 ± 0.16 U/mg protein) 。而固定化海藻糖合成酶最適化反應條件為pH 6.0及50 °C。且固定化後的海藻糖合成酶仍保有良好的熱穩定性,但經過重複批次的使用,固定化海藻糖合成酶會因酵素脫落之問題導致活性下降。

Hydroxyethyl cellulose-coated luffa sponges were prepared by crosslinking Hydroxyethyl cellulose (HEC) onto substrate of luffa sponges as immobilized metal ion affinity absorbent. Covalent linkage of HEC was essential for increasing the surface hydroxyl groups. And then, the HEC -coated luffa sponges were activated by Epichlorohydrin (EPI) , modified by Iminodiacetic acid (IDA) as the chelating agent for loaded with metal ions to attain an immobilized metal affinity absorbents.
The HEC-coated luffa sponges were used for purification and immobilization of the poly(His)-tagged trehalose synthase. The maximum adsorption capacities of trehalose synthase on the HEC-coated luffa sponges were 2.45 mg/g. The Co(II)-loaded adsorbent exhibited the highest specific activity and enzyme activity for the adsorption of the poly(His)-tagged trehalose synthase. The optimal reaction temperature and reaction pH of the immobilized trehalose synthase were identified as 50 °C and pH 6.0. A trehalose conversion yield of 54% was obtained within 4 h at 30 °C and pH 6.0. Both the free and the immobilized enzyme exhibited good thermal stability. A residual activity of approximately 69% 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/11455/91528
其他識別: U0005-1308201517321000
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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