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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/6262


    Title: 鄰苯二酚加氧酵素的熱穩定性提昇研究;Increasing of thermal stability of the aromatic ring cleavage enzyme, catechol 2,3-dioxygenase
    Authors: 梁涵堃;H-K Liang
    Contributors: 生命科學研究所
    Keywords: 熱穩定性;Thermostability
    Date: 2001-07-10
    Issue Date: 2009-09-22 10:16:36 (UTC+8)
    Publisher: 國立中央大學圖書館
    Abstract: 本研究目的在於提昇鄰苯二酚加氧酵素 (catechol 2,3-dioxygenase, C23O)的熱穩定性,並嘗試建立新的酵素熱穩定結構研究模式。所使用之酵素為本土菌株Pseudomonas putida SH1以聚合?鏈鎖反應 (polymerase chain reaction, PCR)方式所得到的C23O基因產物,命名為C23O(SH1)。目前已發現的C23O之最適催化溫度皆為50-60°C,但在此溫度下其熱穩定性只有數分鐘 。至今尚無具良好熱穩定性之苯環切割酵素從自然界中分離出,即使曾報導由嗜高溫的Bacillus thermoleovorans中所分離出之C23O,其在70°C活性半衰期只有1.5 分鐘。1999年Kikuchi等人曾用DNA shuffling的方式取得一具熱穩定性之突變C23O,雖減低了其催化活性,但在50°C的活性半衰期t1/2為70分鐘。本研究則以定位點突變法,依Kikuchi等人之結論改變C23O(SH1)中5 個胺基酸,成功的得到C23O(SH1) N132S/L283T/A284T/K285D/D286Q在50°C活性半衰期 (t1/2)為2.4小時 (提高約2.8倍),同時提高約1.4倍的催化效率。同時以另一株可分解甲苯和二甲苯之P. putida mt-2所選殖出之C23O及其蛋白質的結晶繞射結構 (PDB: 1MPY)為依據,進行同源結構之分子模擬,進行更進一步之結構修飾,嘗試進一步提高C23O(SH1)之熱穩定性。由於近來文獻指出離子鍵為蛋白質熱穩定之關鍵鍵結,故先期研究以離子鍵作為嘗試的模式。在進行過G166D和G166E分別與K170多形成一個表面離子鍵、N271D和N271E分別與K128多形成一個內部離子鍵等離子鍵的增加後,顯示包埋在蛋白內部的離子鍵可能不利於熱穩定,與Kajander等人所推理論相符。而交叉分析的結果顯示如序列的改變視為熱穩定的因子,則因子之間似有量化的加成性關係。未來建議將位於四個次單元中心E223進行可能之包埋電荷的移除,同時建立嗜熱蛋白資料庫以利資料探勘。未來可以C23O快速且靈敏的活性分析法,做為驗證結構熱穩定性演算法之模式。 This research focused on the increasing of the key amino acid residues involved in the thermal stability of an bacteria aromatic ring- cleavage enzyme, catechol 2,3-dioxygenase (C23O). The enzyme, C23O(SH1), was cloned by PCR from a soil bacterium, Pseudomonas putida SH1. The bacterium catabolized phenol, naphthalene, or cresols as sole carbon source to grow. Generally, the optimal temperature of bacterial C23Os are at 50-60°C. But they are only stable for minutes at this temperature range. So far, no thermostable C23O was isolated. The purpose of this study is to make C23O from P. putida SH1 to be more thermal stable at 50oC without interfering activity by site-directed mutagenesis. C23O gene from P. putida SH1 was cloned and expressed in an expression vector pKK223-3 in E. coli JM109 for the mutagenesis experiment. The design of mutagenesis at particular amino acid residues was first based on a previous report on the shuffling of the C23O genes from P. putida G7 and mt-2. A mutant, C23O(SH1) N132S/L283T/A284T/K285D/D286Q , was obtained according the hypothesis of particular regions were critical for thermostability from DNA Shuffling results. The t1/2 was increased to 2.4 hours (About 2.8 Folds). and the catalytic efficiency was increased to 1.4 folds. The known crystal structure of C23O from P. putida mt-2 (PDB: 1MPY) was used as template for the homology modeling of our enzyme for ion pair mutagenesis. The ion pairs critical in thermophilic proteins than mesophilic ones based on crystals in PDB. Because of the complexity of ion pairs in the role of protein thermostablilty, a thermophilic protein database was proposed for further data mining of ion pairs and other structure features in the role of protein thermostability. Several mutated C23Os were generated to manipulate in ion pairs both on surface and inside of the enzyme. Our preliminary mutagenesis of ion pairs lead to unconfirmed conclusion.
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