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標題: 親和性兩水相萃取於蛋白質純化之應用
作者: 黃亮為
關鍵字: 兩水相系統;bovine carbonic anhydrase;親和性萃取;分配係數;抑制劑;熱感性高分子;最低溶解溫度;非共價性鍵結;aqueous two-phase system;affinity ligand;PEG;Dextran;thermosensitive polymer;lower consolute temperature;poly-N-isopropylacryllamide
出版社: 化學工程學系
The partition behavior of bovine carbonic anhydrase(CAB)in aqueous two-phase systems(ATPS)has been examined in order to investigate the effects of changes in phase componenets on the partition coefficient, K. Many factors, including molecule weight of polymers, pH, ionic strength, salt concentration and temperature, can influence partition in aqueous two-phase systems. In this study, the partition of CAB in PEG 4000-Dextran T500 system was performed with three system variables, pH, ionic strength and concentration of NaCl. It was abserved that the partition coefficient of CAB increased with high pH, and decreased the partition coefficient with the addition of NaCl. The inhibitor of CAB, sulfamoylbenzoic acid, was used as an affinity ligand in the ATPS to further enhance the efficiency of separation. The partition coefficient of CAB in the ATPS with inhibitor-conjugated PEG were increased by about twofold. The perspective of substituting PEG with thermosensitive polymer, such as poly-N-isopropylacryllamide(poly-NIPAAm), were also discussed.

Bovine carbonic anhydrase (CAB)在兩水相系統的分配,隨著系統物質的組成以及環境因素的不同,其分配係數,K,也會受到影響。系統的變因主要有pH值、高分子分子量、離子強度、鹽類濃度以及溫度。本研究是使用PEG 4000與Dextran T500的兩水相系統來萃取CAB,我們所探討的因素主要有三個:pH值、離子強度與NaCl的濃度。CAB的分配係數會隨著pH的提高而增加,而NaCl的添加反而會降低分配效果。
親和性萃取也常應用於兩水相系統,最常被當作親和性基之載體的高分子為PEG,主要由於其使用性廣泛以及具有亦反應的官能基。本研究採用CAB的抑制劑,sulfamoylbenzoic acid作為本系統的親和性基。其與CAB具有專一性的吸引力,因此抑制劑在與PEG反應鍵結後,加入兩水相系統中,K值約可提高2~3倍。
之後,再以熱感性高分子,poly-N-isopropylacryllamide (poly-NIPAAm)代替PEG,由於poly-NIPAAm的LCT很低,在37 oC左右即可沈澱,有利於與CAB的分離以及高分子回收。比較PEG-Dextran 以及 poly-NIPAAm-Dextran兩個系統萃取效果,我們期望能找到最適化的分離條件以及最佳的生物分離程序。
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