Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/99522
標題: 以空氣凝膠固定化金屬親和吸附基材進行純化及固定化過程
Aerogels-based immobilized metal affinity chromatography adsorbents for enzyme purification and immobilization
作者: 章譽騰
Yu-Tung Chang
關鍵字: 纖維素;空氣凝膠;海藻糖合成酶;蛋白質純化;酵素固定化;cellulose;immobilized enzyme;protein purification
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摘要: 
本研究空氣凝膠當作固定化金屬親和吸附材的載體,再依序接上環氧氯丙烷(EPI)及金屬螯合劑亞胺二乙酸(IDA),最後再接上過渡金屬離子,以上部份即完成固定化金屬吸附之基材。
完成後的金屬螯合親和吸附基材的過渡金屬離子可以與含有His-tag基因重組海藻糖合成酶的His-tag形成配位共價鍵,以達到想要的純化及固定化海藻糖合成酶的目的。
在此次研究結果中,得到的最適化的IDA濃度與時間比例為1M:10小時,也就是銅離子鍵結量為1130.7 μmol/g的情況下,比其他比例的基材吸附效果還要好。由Langmuir model 可以得知金屬螯合親和吸附材最大吸附量為0.52 mg/g以及解離常數為0.069 mg/ml。比較在不同的條件下的鎳離子固定化之後的海藻糖合成酶,以IDA濃度與時間比例為1M:10小時的條件下所呈現的總活性與比活性最高。
完成以共價鍵結固定之固定化酵素進行重複批次操作研究,重複操作的效果在經過20次的操作之下,其相對活性大約下降17% 左右,未見其明顯的下降。
綜合以上結果,確定了系統整合純化以及固定化的可行性,並且此固定化是以共價鍵結固定化,在操作上比較不會產生有酵素脫落的情況。

In this study, Aerogel-based immobilizied metal affinity chromatography adsorbents with ligand densities were prepared for the purification and immobilization of poly(His)-tagged proteins. The adsorbents were activated by Epichlorohydrin (EPI) , modified by Iminodiacetic (IDA) as the chelating agent for loaded with metal ions.
The complete transition metal ions can form a coordinate covalent bond with the His-tag containing the His-tag gene recombinant trehalose synthase enzyme,in order to achieve the desired purpose of purification and immobilization of trehalose synthese enzyme.
In this study, the optimum IDA concentration to time ratio obtained was 1 M:10 hours, that is,the copper ion binding capacity was 1130.7 μmol/g, which was better than that of other substrates. The Langumuir model shows that the maximum adsorption capacity of the metal chelate affinity adsorbent is 0.52 mg/g and the dissociation constant is 0.069 mg/ml. Comparing the nickel ion immobilizied trehalose synthase under different conditions, the total activity and the specific activity exhibited IDA concentration and time ratio of 1M: 10 hours conditions by the highest.
Completion of the covalent immobilization of immobilized enzyme batch operation study, the effect of repeated operations after twenty times the operation, the relative activity fell 17% about no significant drop.
Based on the above results, the feasibility of system integration, purification and immobilization was confirmed, and the immobilization was immobilization by covalent bonding, so that enzyme shedding would not occur in operation.
URI: http://hdl.handle.net/11455/99522
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