| Summary: | 表面表現蛋白質時，目標蛋白在誘導期間長時間暴露於培養環境中，在高轉速環境下，環境中的剪力可能造成蛋白質變性。本研究探討在不同培養條件下，培養轉速、培養的體積、容器的樣式等因素對於表面表現目標蛋白(surface-displayed Candida antarctica lipase A, sdCALA)之比活性影響，同時以動力學角度分析不同轉速對其動力學特性之影響，最後以最佳培養條件培養表現sdCALA，並分析其熱穩定性及有機溶劑耐受性。 實驗結果發現，在轉速為50 rpm，其比活性受氧氣濃度限制，比活性不高；在100 rpm以上，其比活性主要受剪力影響；在培養體積研究方面，Erlenmeyer型搖瓶培養體積對剪率的影響較小，體積主要影響質傳係數KLa，因此也影響比活性表現；Hinton型搖瓶與Erlenmeyer型搖瓶狀況類似，但以50 ml培養體積表現較低的比活性，經計算後發現應是受剪率影響。Hinton培養比Erlenmeyer培養有較佳的菌量生長，以100 ml培養體積於500 ml Hinton培養sdCALA有最佳比活性(0.243±0.020 U/mg)。在最適溫度及pH的研究方面，其最適溫度及pH為60˚C及pH 6，且培養轉速對sdCALA之最適溫度與pH無影響。在動力學分析方面，以最佳培養條件培養之sdCALA較以200 rpm培養之sdCALA以及胞內表現之CALA有更高之VMax及較低之Km，分別為0.070 μmole ml-1 min-1及0.087 μmole ml-1。在熱穩定性方面，sdCALA在70˚C下培養30分鐘後仍殘餘80 %以上活性，在80˚C以上活性迅速受溫度影響而下降；在有機溶劑耐受性方面，在四種親水性有機溶劑中，以總濃度30 %乙醇有最高之活性為0.67 U/ml；以異丙醇為溶劑，其結果與乙醇類似，在20%濃度下有最高之活性(0.51 U/ml)；以乙腈、丙酮為溶劑，不論何種濃度下其活性皆不高。 For a surface-displayed protein, the target protein is exposing directly to the culture environment during the induction period. The shaking speed of culture condition may cause the shear-denaturation. In this study the culture conditions (shaking speed, working volume, flask types) were tested to observe their influence on the surface- displayed protein (i.e., Candida antarctica lipase A, CALA). The specific activities of sdCALA cultivated under 50 rpm were limited by the dissolved oxygen level regardless the flask types. When the shaking rate was above 100 rpm, the specific activities were mainly influenced by the shear stress. For Erlenmeyer flasks, the working volume significantly affects oxygen mass transfer rate (KLa), which in turn influences the specific activities of sdCALA. The results of Hinton flasks were similar to Erlenmeyer except the 50 ml sample, which showed a lower specific activity. This result was probably caused by the high shear rate based on our calculation. The optimal culture condition was found as follows: 500 ml Hinton flask containing 100 ml of medium with the shaking rate of 100 rpm, where the optimal sdCALA specific activity was 0.243±0.020 U/mg. For the temperature and pH effect, the optimal activity was observed at 60˚C and pH 6. The sdCALA obtained under various shaking rate gave the same temperature and pH profiles. The kinetic study revealed that the sdCALA obtained under optimal condition gave a higher VMax (0.070 μmole ml-1min-1) and a lower Km (0.087 μmole ml-1) than that of the sdCALA obtained under 200 rpm and the free CALA. For the thermal stability test, the sdCALA retains over 80 % of activity at 70˚C for 30 minutes and the activity was rapidly decreased when the temperature is over 80˚C. Four different hydrophilic organic solvents were used for the organic solvents tolerance test. The highest activity (0.67 U/ml) was obtained when the ethanol concentration was set at 30%. For isopropanol, the result was quite similar to that of ethanol. sdCALA performed best under 20% concentration (0.51 U/ml). When acetonitrile and acetone are present, sdCALA performed worse regardless of the concentrations. 中文摘要 i Abstract ii 圖目錄 v 表目錄 vi 縮寫字對照表 vii 第一章、緒論 1 1.1 前言 1 1.2研究動機 2 第二章、文獻回顧 3 2.1 脂肪酶(Lipase) 3 2.1.1 簡介 3 2.1.2 Candida antarctica lipase A 5 2.2細胞表面表現系統(Cell surface display system) 7 2.3 冰核蛋白(Ice nucleation protein) 10 2.4 攪拌及溶氧等相關計算 11 第三章、實驗材料與方法 13 3.1實驗材料 13 3.1.1 實驗儀器設備 13 3.1.2實驗藥品 15 3.1.3 實驗用溶液 16 3.2實驗流程 19 3.3實驗方法 21 3.3.1 菌株與質體 21 3.3.2菌種保存與培養方法 25 3.3.3 破菌方法 26 3.3.4 CALA活性分析 (Activity assay) 26 3.3.5 溫度效應 28 3.3.6 pH效應 28 3.3.7 動力學研究(kinetic study) 29 3.3.8 溫度穩定性測試 29 3.3.9 有機溶劑耐受性測試 29 3.3.10 黏度測量 30 第四章、結果與討論 31 4.1 培養轉速對比活性之影響 31 4.2 培養體積對比活性之影響 35 4.3 CALA及sdCALA最適溫度探討 38 4.4 CALA及sdCALA最適pH探討 40 4.5 CALA及sdCALA動力學分析 42 4.6 sdCALA熱穩定性分析 46 4.7 sdCALA耐有機溶劑分析 47 第五章、結論與未來展望 49 5.1 結論 49 5.2 未來展望 50 參考文獻 51
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