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標題: 金屬親和吸附材於蛋白質純化及酵素固定化之應用
作者: 何立凡
關鍵字: IMAC
His-tagged protein
protein purification
enzyme immobilization
出版社: 化學工程學系
摘要: 本研究利用自行合成的金屬親和吸附材將酵素(D-hydantoinase)純化及固定於吸附基材上,並直接應用於連續式反應操作希望建立一個可以同時純化及固定化酵素並可確實應用於反應操作的系統。 實驗材料選取矽膠為基材,因其表面具有OH基所以易於化學改質且機械強度夠,菌種取自於中興大學分生所許文輝教授成功發展的基因重組E.coil,利用基因重組技術,使E.coil所生產D-hydantoinase尾端均帶有六個組氨酸(histidine),如此即可以金屬親和吸附法純化之。 實驗結果發現雖然純化的效果不若商用純化膠體(Ni-NTA)來的好,且自行合成的吸附材上吸附較多的雜蛋白質,但在反應操作卻得到不錯的結果。不僅沒有其他的副反應產生且固定後的酵素最適反應溫度與pH值亦提昇。
Several silica-based immobilized metal affinitychromatography (IMAC) adsorbents with different ligand densities(3-Glymocidoxyprop- -yltrimethoxysilane-Iminodiacetic acid to Iminodiacetic) were prepared for the purification of proteins and immobilization of enzymes. The IM- -AC adsorbents prepared with a ratio of 1:5 exhibit optimal binding cap- acity for His-tagged proteins and minimal nonspecific adsorption. Results of SDS-PAGE indicate that the adsorbent can be used for the purification of His-tagged D-hydatoinase(DHTase) for crude cell lysate in a single step process with a purify above 90%, comparable to commercial agarose -based IMAC adsorbents. Due to it's high mechanical strength, the silica- -based IMAC adsorbents are particularly well suited for industrial operations. A packed-bed bioreactor with the aforementioned IMAC adsorbent,loaded with DHTase was used forthe continuous bioconversion of D,L-p-HPH to N-carbamoyl-D-hydroxyphenylglicine. Under optimal conditions, 60℃,pH 8,a conversion of 60% was observed at a residue time of 20 mins. In this study ,we successful demonstrate that a packed- -bed bioreactor containing silica-based IMAC adsorbents can be employed for the purification and immobilization of His-tagged enzymes.
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