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標題: 聚甲基丙烯酸二羥基乙酯固定化金屬親和吸附材於蛋白質純化之應用
作者: 劉哲良
關鍵字: 聚甲基丙烯酸二羥基乙酯;固定化金屬親和吸附材
出版社: 化學工程學系
In this study, polyhydroxymethacrylate ( pHEMA ) microbeads were prepared by a suspension polymerization technique and modified by IDA( iminodiacetic acid ), loading with Ni2+, and used as adsorbent to purify a target protein (D-hydantoinase , DHTase) from the large volumes of liquid culture medium. In the text, the such are factors that influence the adsorption of DHTase are discussed, for example: category of metal ion, crosslinking ratio (EGDMA to HEMA) , pH ionic,salt composition, and NaCl conc. in the buffer. The results indicate that the optimum adsorbent crosslinking ratio is 2.0/30 and water content is 46%. The optimum purification procedure condition is 4℃、0.5M NaCl、pH 6.0、50mM Tris-HCl buffer for loading, and 4℃、300mM NaCl、300mM Imidazole、pH 8.0、50mM phosphate buffer for elution, which results in a total protein adsorption incrase from 3.9 mg g-1 (under non-optimal condition) to 7.8 mg g-1, a total protein elution increase from 2.39 mg g-1 to 4.33 mg g-1, and a purification of 91%. After seven repeated uses, metals ion was loss about 30%, and total protein elution also declines to 50%. The adsorption phenomena appeared to follow a typical Langmuir isotherm. The maximum capacity (Qm) of the Ni2+ -complexed beads for D-hydantoniase is 10.17 mg g-1, and the dissociation constant (kd) is 1.12*10-2 g ml-1.

本研究以懸浮聚合法技術,自行合成高分子~聚甲基丙烯酸二羥基乙酯(pHEMA)微珠作為固體載體,利用化學合成的方式接上IDA(iminodiacetic acid)螯合劑,作為固定化金屬親和吸附材,並利用所得之金屬親和吸附材用於純化粗醱酵液中之右旋乙內醯尿脢(D-hydantoinase,簡稱DHTase)。文中探討各種影響DHTase吸附量及純化的原因,例如:金屬離子的種類、高分子之交聯比例、pH值、各種鹽類及NaCl的濃度等等。經由實驗所得到結果,最適化的高分子交聯比例為30/2.0 (HEMA/EGDMA),含水率約為46%,並螯合鎳離子。而吸附的最適化條件為:溫度4℃、0.5M的NaCl、pH 6.0、50mM Tris-HCl buffer狀態下最佳。而脫附的最適化條件為:溫度4℃、300mM NaCl、300mM Imidazole、pH 8.0、50mM的磷酸緩衝溶液狀態下為最佳。經由上述最適化條件,總蛋白質吸附量會由(未添加鹽類及改變pH值)3.9(mg/g-gel)增加到7.8(mg/g-gel),總蛋白質脫附量也由2.39(mg/g-gel)增加到4.33(mg/g-gel),純度為91%。膠體重複使用七次後,金屬離子掉落約30%,總蛋白質脫附量也降到50%左右。利用Langmuir equation並假設蛋白質與配位基間為單一層均勻吸附作用,獲得動力參數:最大吸附量(Qm)為10.17(mg/g-gel),游離常數(kd)為1.12*10-2(g/ml)。
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