- 學位論文
澱粉分解酶之熱穩定性與其復性難易之關係
The relationship between thermal stability and refolding process of α-amylases
摘要
本研究利用Bacillus amyloliquefaciens α-amylase(BAA)及Bacillus licheniformis α-amylase(BLA)兩種a-amylase，觀察在不同之溫度下，BAA與BLA隨溫度變化其結構與活性變化之情形。實驗中利用圓極偏光光譜觀察二級結構的破壞情形；以螢光光譜觀察其內胺基酸tryptophan的暴露情形；以活性分析觀察活性破壞的情形，並求出BAA與BLA受熱失活過程中熱力學性質的變化。藉此我們可以觀察BAA與BLA受熱失活過程中之異同，並與加入變性劑變性的結果加以比較。另一方面，我們使用胍鹽酸使α-amylase展開變性，試圖藉由butyl-CM-sepharose協助BAA與BLA復性，來觀察此兩種α-amylase摺疊過程中的異同。 實驗結果發現BAA與BLA隨溫度上升，其結構與活性有逐漸被破壞之現象，且BLA之二級結構較BAA穩定。對BAA而言，其結構一旦被破壞，也會立刻喪失其活性，而BLA則是疏水區域先暴露，接著才是二級結構與活性破壞。而由熱力學性質所得之結果發現，BLA之三級結構回復速度較慢。 在復性實驗中，經由butyl-CM-sepharose的幫助，原本以直接稀釋法，需在濃度降至0.003 mg/mL以下才能復性的BAA，已能在濃度0.56 mg/mL下獲得70%的活性回收，而以直接稀釋法無法復性的BLA，也能在濃度0.45 mg/mL下，回收38%的活性。 經比較BAA與BLA之復性結果發現butyl-CM-sepharose僅幫助BAA及BLA部分摺疊，而部分摺疊的蛋白質仍會產生糾結。BLA的質量與活性回收率均低於BAA。由BAA與BLA受熱失活的結論得知，BLA復性結果較不理想的原因應為BLA的三級結構回復速度較慢所導致。
並列摘要
The main purpose of this study is to study how the secondary and tertiary structure changes relate to the protein stability and refolding. The unfolding and refolding processes of two a-amylases derived from Bacillus licheniformis (BLA) and Bacillus amyloliquefaciens (BAA) were investigated and compared. The thermal stability of a-amylase was monitored by the loss of its secondary structure and tertiary structure, as well as the activity loss. The loss of (a-helix was chosen to represent the secondary structure change and was monitored under circular dichroism. The percentage of (a-helix loss at various temperatures could be estimated. The tertiary structure was symbolized by the exposure of a hydrophobic amino acid, tryptophan, and could be monitored by the fluorescence spectroscopy. The kinetics and the thermodynamics of the disruption of a-amylase’s secondary and tertiary structure could therefore be monitored. It was found that, for either BAA or BLA, the destruction of the secondary structure proceeded in a rate similar to that of activity loss. However, the destruction of the secondary structure of BLA proceeded in a rate much slower than that of its tertiary structure. The results indicated that the activity stability of a-amylases depend more on the stability of their secondary structure than their tertiary structure and the superior thermal stability of BLA origins from its stronger secondary structure. Both a-amylases were not refolded by direct dilution at a final concentration larger than 0.003 mg/mL. By the assistance of a self-synthesized Butyl-CM-sepharose particle, the activity recovery of BAA reached 70% at a final concentration of 0.56 mg/mL and the activity recovery of BLA reached 38% at a concentration of 0.45 mg/mL. It was suspected that both a-amylases were only partially refolded on Butyl-CM-sepharose. The further refolding of BLA was slower than that of BAA so that the activity recovery of BLA was lower.