臺灣博碩士論文加值系統

雙水相系統（Aqueous Two-Phase System, ATPS）已被廣泛應用於生物材料的分離
過程。 ATPS 的優點為低界面張力，良好的生物相容性和產率。雙水相系統影響參數
會由pH 值，節線長度（Tie-line, TLL），雙節曲線，離心速度，工作溫度，離子強度
和體積比的不同分離結果有所差異。本研究使用的含水兩相系統（ATPS）溶液進行，
以回收所表達的蛋白酶從鳳梨粗取液調查純化。
本研究利用鳳梨果肉(Ananas comosus Merr. Tainung No 17)動力攪拌成粗取液，接著
以雙水相系統分離目標物鳳梨蛋白酶，找出最適化分離條件。利用磷酸鹽系統及檸檬
酸鹽系統測試不同相圖之節線長度，比較鳳梨蛋白酶之分配係數及純化倍率，進行標
準品及粗取液(Crude extract)雙水相系統分離，選定磷酸鹽系統加以進行環境條件之參
數操作。最佳結果為包含選擇不同環境pH 值、雙結點曲線(Binodal curve)、節線長度
(Tie-line length, TLL)、分離速度(Centrifugation speed)、鹽類(Salt)對鳳梨蛋白酶的影響，
進行分離效果的探討。目前最適化分離結果由粗取液經由17% (w/w)的PEG 4000 /14%
(w/w) 的PO4 pH 8 所組成於分離速度8000×g 室溫操作下分離後，其活性為2.7 U、純
化倍率(PF)達16.3 及回收率可達55.6%。研究結果顯示雙水相系統是一高效率的蛋白
純化及分離技術，可直接由鳳梨粗取液中分離出鳳梨蛋白酶。

Aqueous two-phase system (ATPS) has been widely applied in various separation
processes of biomaterials. ATPS possesses several advantages, such as low interfacial
tension, good biocompatibility and resulting in high yield of target product. The partition
behaviour of a target product in an ATPS could be affected by tie-line length (TLL),
centrifugation speed, operational temperature, ionic strength, pH and volume ratio (VR) of
the ATPS. In this study, recovery of expressed protease from flesh pineapple fruit using
polymer/salt ATPS was investigated. The partition behaviour of bromelain standard in the
developed polyethylene glycol (PEG)/phosphate ATPS was also studied. Screening of the
partition behaviour of bromelain by PEG/Phosphate/Citrate systems indicated that
(PEG)/phosphate system showed the better partition coefficient and purification. The
operating factors includes pH value, binodal curve, tie-line length (TLL), centrifugation
speed, ionic strength, and operational temperature upon recovery of bromelain from
pineapple using PEG/phosphate ATPS were investigated and discussed. The results
demonstrated that the optimal conditions for bromelain recovery from crude extract were
obtained at ATPS consisted of 17% (w/w) PEG 4000 and 14% (w/w) PO4 at pH value of 8,
centrifugation speed of 8000×g ；and at room temperature. The resulting specific activity
and purification factor (PF) of bromelain at top phase of PEG/phosphate ATPS were 2.7 U
and 16.3 respectively. The efficiency of recovery achieved 55.6%. The findings illustrated
that ATPS could be a promising technique to recover bromelain in one-step operation.

目錄
摘要 ......................................................................................................................................... 2
Abstract ................................................................................................................................... 3
致謝 ......................................................................................................................................... 4
目錄 ......................................................................................................................................... 5
圖目錄 ..................................................................................................................................... 9
表目錄 ................................................................................................................................... 11
第一章 緒論 .......................................................................................................................... 12
1.1 前言 .......................................................................................................................... 12
1.2 蛋白質純化技術 ...................................................................................................... 13
1.2.1 膠體過濾技術 ............................................................................................... 14
1.2.2 離子交換層析技術 ....................................................................................... 15
1.2.3 疏水性層析技術 ........................................................................................... 16
1.2.4 親和性層析技術 ........................................................................................... 17
1.2.5 雙水相系統 ................................................................................................... 18
1.2.5.1 雙水相系統的特性 ............................................................................ 19
1.2.5.2 雙水相系統相圖 ................................................................................ 20
1.2.5.3 雙水相系統分類 ................................................................................ 22
1.2.5.3.1 聚合物—鹽類系統 ................................................................. 24
1.2.5.3.2 聚合物—聚合物系統 ............................................................. 24
1.2.5.3.3 離子溶液系統 ......................................................................... 24
1.2.5.4 雙水相系統的分配係數(K) .............................................................. 24
6
1.2.5.5 雙水相系統的分析 ............................................................................ 26
1.3 蛋白酶 ...................................................................................................................... 27
1.3.1 蛋白酶簡介 ................................................................................................... 27
1.3.2 蛋白酶分類 ................................................................................................... 30
1.3.3 鳳梨蛋白酶簡介 ........................................................................................... 33
1.3.4 鳳梨蛋白酶應用 ........................................................................................... 34
1.4 研究目的與策略 ...................................................................................................... 35
1.4.1 研究目的 ....................................................................................................... 35
1.4.2 研究策略 ....................................................................................................... 36
第二章 以不同雙水相系統組成分離純化鳳梨粗取液之鳳梨蛋白酶 .............................. 37
2.1 導論 .......................................................................................................................... 37
2.1.1 鳳梨蛋白酶純化之技術及研究 .................................................................. 37
2.2 實驗藥品 .................................................................................................................. 39
2.3 實驗儀器設備 .......................................................................................................... 40
2.4 實驗方法與分析 ...................................................................................................... 41
2.4.1 鳳梨蛋白酶製備 ........................................................................................... 41
2.4.2 鳳梨蛋白酶活性測定 ................................................................................... 41
2.4.3 聚丙烯醯胺膠體電泳分析 ........................................................................... 41
2.4.4 Coomassie blue 呈色法 ............................................................................... 43
2.4.5 總蛋白質濃度測定 ....................................................................................... 44
2.4.6 雙水相系統製備 ........................................................................................... 44
2.4.8 雙水相系統之雙節點曲線建立 ................................................................... 45
2.5 結果與討論 ............................................................................................................. 47
2.5.1 探討不同系統組成之雙節點曲線對ATPS 分離效果影響 ...................... 47
7
2.5.2 探討系統組成對鳳梨蛋白酶分離效果影響 .............................................. 51
2.5.3 利用SDS-PAGE 進行ATPS 純化結果之分析 ........................................... 52
2.6 結論 ......................................................................................................................... 53
第三章 雙水相系統參數操作之最適化條件探討 .............................................................. 54
3.1 導論 ......................................................................................................................... 54
3.2 實驗藥品 ................................................................................................................. 54
3.3 實驗儀器設備 ......................................................................................................... 54
3.4 實驗方法與分析 ..................................................................................................... 55
3.4.1 鳳梨蛋白酶製備 ........................................................................................... 55
3.4.2 鳳梨蛋白酶活性測定 .................................................................................. 55
3.4.3 總蛋白質濃度測定 ...................................................................................... 55
3.4.4 雙水相系統之參數操作 .............................................................................. 55
3.4.4.1 系統組成之影響 ............................................................................... 55
3.4.4.2 pH 值之影響 ...................................................................................... 56
3.4.4.3 鹽類添加之影響 ............................................................................... 56
3.4.4.4 分離速度之影響 ............................................................................... 57
3.4.4.5 不同體積比及溫度之影響 ............................................................... 57
3.5 結果與討論 .............................................................................................................. 58
3.5.1 探討雙節點曲線對ATPS 分離效果影響 ................................................... 58
3.5.2 探討pH 值對ATPS 分離效果影響 ............................................................. 60
3.5.4 探討鹽類對ATPS 分離效果影響 ............................................................... 61
3.5.5 探討不同分離速度對ATPS 分離效果影響 .............................................. 63
3.5.6 探討不同體積比及溫度對ATPS 分離效果影響 ...................................... 65
3.5.7 利用SDS-PAGE 進行ATPS 純化最適化結果之分析 ............................... 67
8
3.6 結論 ......................................................................................................................... 68
第四章 總結與未來工作 ...................................................................................................... 69
4.1 總結 .......................................................................................................................... 69
4.2 未來工作 .................................................................................................................. 69
參考文獻 ............................................................................................................................... 70
圖目錄
圖1-1、膠體過濾技術原理圖.............................................................................. 14
圖1-2、離子交換技術原理圖解 ......................................................................... 15
圖1-3、疏水性層析技術原理圖解 ..................................................................... 16
圖1-4、親和性層析技術原理圖解 ..................................................................... 17
圖1-5、雙水相系統萃取原則流程圖(Hustedt, 1986) ........................................ 18
圖1-6、雙水相系統相圖圖示(Hatti-Kaul, 2000) ................................................ 21
圖1-7 研究架構圖 ................................................................................................ 36
圖2-1 PEG 2000 之折射率檢量線 ...................................................................... 45
圖2-2 PEG 4000 之折射率檢量線 ...................................................................... 45
圖2-3 PEG 6000 之折射率檢量線 ...................................................................... 45
圖2-4 PEG 8000 之折射率檢量線 ...................................................................... 45
圖2-5 Potassium phosphate pH7.5 之折射率檢量線 ........................................... 46
圖2-6 Potassium phosphate pH7.5 之電導度檢量線 ........................................... 46
圖 2-7 PEG 8000 / PO4 於 pH 7.5 的 Binodal curve ............................................. 48
圖 2-8 PEG 6000 / PO4 於 pH 7.5 的 Binodal curve ............................................. 48
圖2-10 以SDS-PAGE 分析標準品及粗取液經雙水相系統純化之結果 .......... 52
圖3-1 PEG 4000 / PO4 於pH 7.5 的 Binodal curve ...................................... 58
圖3-2 不同TLL 對標準品中鳳梨蛋白酶分配係數的影響 .............................. 59
圖3-3 不同TLL 對粗取液中鳳梨蛋白酶分配係數的影響 .............................. 59
圖3-4 pH 值對標準品中鳳梨蛋白酶分配係數的影響....................................... 60
圖3-5 pH 值對粗取液中鳳梨蛋白酶分配係數的影響....................................... 61
10
圖3-6 添加鹽類對標準品中鳳梨蛋白酶分配係數的影響 ............................... 62
圖3-7 添加鹽類對粗取液中鳳梨蛋白酶分配係數的影響 ............................... 63
圖3-8 不同分離速度對標準品中鳳梨蛋白酶分配係數的影響 ....................... 64
圖3-9 不同分離速度對粗取液中鳳梨蛋白酶分配係數的影響 ....................... 64
圖3-10 ATPS 中不同體積比在300 K 下對鳳梨蛋白酶分配係數之影響 ........ 66
圖3-11 ATPS 中不同體積比在277 K 下對鳳梨蛋白酶分配係數之影響 ........ 66
圖3-12 以SDS-PAGE 分析粗取液經雙水相系統最適化結果之分析 .............. 67
11
表目錄
表 1-1 依據蛋白質不同特性做分離分類 ........................................................ 13
表1-2、不同種類的雙水相系統 ......................................................................... 23
表1-3 存在自然界中的蛋白酶 (Adler - Nissen, 1986) ..................................... 28
表2-1 不同純化技術之產率及純化倍率 ........................................................... 38
表2-2 實驗所使用之藥品.................................................................................... 39
表2-3 實驗所使用之儀器設備............................................................................ 40
表2-4 Gel formulation for SDS-PAGE analysis of protein ................................... 42
表2-5 Running buffer (1X) ................................................................................... 43
表2-6 磷酸緩衝溶液(Phosphate buffered saline, PBS) ...................................... 43
表2-7 樣品緩衝液(Sample buffer) (2X) .............................................................. 43
表2-8 染色液(Staining solution) .......................................................................... 44
表2-9 去色液(De-staining solution) .................................................................... 44
表 2-10 PEG/Sodium citrate 和PEG/Phosphate 系統不同分子量及TLL 之Ke
和PFT 比較 .................................................................................................... 50
表 2-11 系統組成對粗取液及標準品分配係數比較 ......................................... 51
表3-1 不同節線長度之系統組成 ....................................................................... 55
表3-2 不同pH 值之磷酸鉀鹽溶液配製 ............................................................. 56

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