臺灣博碩士論文加值系統

本研究探討以氫氧基磷灰石做為固定化金屬親和吸附材及其於基因重組 D-hydantoinase(DHTase)純化之應用。最適回收純化程序: 配位金屬離子為Fe3+，吸附緩衝液 20mM Tris-HCl 0.25M NaCl 0.05M NaF pH 7.5，清洗緩衝液0.1M sodium phosphate pH 7.0，脫附緩衝液0.15M sodium phosphate pH 8.0。在最適條件時，總蛋白質吸附量為9.92 mg/g HAP，D-hydantoinase 吸附量為4.9 mg /g HAP，活性回收率為40%，純度為≧95%。研究結果顯示，以Fe3+為配位金屬離子有最佳的吸附選擇性，pH 7.5可以提升酵素回收率與活性，氯化鈉與氟化鈉的添加可以減少非特定吸附產生與增加回收率。由2-epimerase的吸附則證明了此吸附材於基因重組蛋白質的吸附再現性，以及吸附純化程序於失活條件下亦可進行。

In this study, the application of hydroxyapatite as IMAC absorbent for the purification of soluble recombinant poly(His)-tagged D-hydantoinase and 2-epimerase was investigated. Fe3+ as the coordinating metal ion showed the best adsorption selectivity. The addition of 0.15~0.25M NaCl and 0.05M NaF can increase D-hydantoinase recovery and activity. Changes in pH and concentration of sodium phosphate buffer can wash nonspecifically adsorbed proteins and elute D-hydantionase from adsorbent effectively. The recovery yield and purity of D-hydantoinase were 40% and above 95% respectively, under optimal purification conditions. The capacity of hydroxyapatite-based IMAC absorbent was 9.92 mg/g HAP for total proteins, and 4.9 mg/g HAP for D-hydantoinase, The absorption of denatured 2-epimerase demonstrate that hydroxyapatite-based IMAC absorbent can be applied to purification of recombinant poly(His)-tagged protein under denaturing conditions.

第一章 緒論....................................................8
第二章 文獻回顧................................................9
2-1 蛋白質純化.................................................9
2-2 蛋白質純化策略............................................12
2-3 蛋白質純化單元............................................14
2-4 固定化金屬離子親和法......................................17
2-5 吸附條件的探討............................................19
2-5-1 過渡金屬離子........................................19
2-5-2 pH..................................................20
2-5-3 鹽類................................................21
2-5-4 清洗與脫附條件......................................22
2-6 研究系統..................................................23
2-6-1 D-hydantoinase......................................23
2-6-2 氫氧基磷灰石........................................24
第三章 實驗藥品與儀器.........................................27
3-1 實驗藥品..................................................27
3-2 實驗儀器..................................................28
第四章 實驗方法...............................................30
4-1 菌種的培養................................................30
4-1-1 LB固態培養基的製備..................................30
4-1-2 LB液態培養基的製備..................................31
4-1-3 五公升桌上型發酵槽操作..............................31
4-2 菌體的回收與粗酵素液製備..................................32
4-2-1 菌體的回收..........................................32
4-2-2 粗酵素液製備........................................32
4-3 分析方法..................................................32
4-3-1 蛋白質濃度分析......................................32
4-3-2 SDS-PAGE 電泳分析...................................33
4-3-3 D-hydantoinase 活性分析.............................35
4-3-4 TINA影像分析........................................36
4-4 批次吸附實驗..............................................37
4-5 D-hydantoinase最適純化程序探討............................39
4-5-1 金屬離子最適化......................................39
4-5-2 吸附pH最適化........................................40
4-5-3 鹽類最適化..........................................41
4-5-3-1 氯化鈉效應..................................41
4-5-3-2 氟化鈉效應..................................42
4-5-3-4 固定氟化鈉濃度探討氯化納之效應..............45
4-5-4 清洗(Wash)與脫附(Elute)條件的探討...................46
4-5-4-1 利用二氮二烯五圜為清洗與脫附主要組成........46
4-5-4-2 以磷酸納取代二氮二烯五圜做為清洗緩衝液之組成47
4-5-4-3 以磷酸鈉為清洗及脫附緩衝液之組成............48
4-6 磷酸鈉清洗與脫附緩衝液系統之吸附數據探討..................49
4-6-1 總蛋白質與D-hydantoinase吸附量探討..................49
4-6-2 活性回收率及純化因子之探討..........................50
4-6-3 最適純化產物純度分析................................51
4-7 2-epimerase的吸附.........................................51
4-7-1 不可溶性2-epimerase之吸附...........................52
4-7-2 可溶性2-epimerase之吸附.............................53
第五章 結果與討論.............................................54
5-1 D-hydantoinase最適純化程序探討............................54
5-1-1 金屬離子最適化......................................54
5-1-2 吸附pH最適化........................................56
5-1-3 鹽類效應............................................57
5-1-3-1 氯化鈉效應..................................58
5-1-3-2 氟化鈉效應..................................59
5-1-3-3 固定氯化鈉濃度探討氟化鈉效應................60
5-1-3-4 固定氟化鈉濃度探討氯化鈉效應................63
5-2 清洗與脫附條件的探討......................................64
5-3 2-epimerase的吸附.........................................66
第六章 結論...................................................67
第七章 參考文獻...............................................70

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