果糖轉移酶的純化和特性分析及定點突變研究

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標題:	果糖轉移酶的純化和特性分析及定點突變研究 Purification and Characterization of fructosyltransferases and site-directed mutagenesis studies
作者:	謝智宇
貢獻者:	嘉南藥理科技大學：生物科技系暨研究所江建民
關鍵字:	果醣轉移酶突變蛋白質純化 protein purification mutation. fructosyltransferases
日期:	2009
上傳時間:	2011-10-27 14:43:16 (UTC+8)
摘要:	果糖轉移酶 (Fructosyltransferases; FT) 的主要催化蔗糖身上果糖基轉移到另一個蔗糖或用來生成果糖鏈。此類酵素根據其序列被分類屬於醣苷鍵水解酶第32號家族的蛋白質，這個家族已知為一種雙結構區的酵素：N端結構為5個β折板所形成的螺旋槳結構，C端為由β折板所形成的三明治結構。對Aspergillus japonicus純化出的AjFT進行特性分析，其分子量大小約為100KDa，其中醣基化修飾約占30KDa，在水溶液中是以3聚體形式存在。酵素的活性分析結果顯示，此酵素在15分鐘反應時間條件下，70℃是其最高效率的反應溫度，pH 6.0有最佳的反應活性。在60℃以上，酵素的活性會隨著時間延長或溫度的提升而減少。對於其結構的穩定性檢測發現3%的SDS對此酵素的活性不會有影響，但1M的Guanidine-HCl及3.5M Urea則會使酵素活性喪失一半。目前取得的13株針對異源基因表達AjFT的定點突變突變株，經過初步的活性檢測發現，突變活性區周圍的胺基酸將導致酵素活性有不同程度的改變，其中大部分是降低酵素的轉移活性，也有一些突變會使得活性有所上升。這些位置的影響在本論文中被討論。 Fructosyltransferases (FTs) are enzymes that catalyze the transfer of the fructosyl residue from sucrose to another sucrose molecule or cause a growing fructan chain. FTs belong to glycoside hydrolase family 32 according to the sequence similarity. The enzymes of this family were reported to contain two domains: the N-terminal domain consisting of five bladed β-propeller fold connected to the C-terminal domain which presents a β-sandwich fold. A fructosyltransferase was purified from Aspergillus japonicus and its activity was characterized. The molecular weight of the purified enzyme estimated by SDS-PAGE was 100 KDa, containing about 30 KDa glycosyl part, as compared to its theroretical molecular weight. The enzyme shows an optimal reaction temperature at 70℃, and an optimal pH at 6.0. The higher the temperature or the longer the preincubation time, the enzyme lost more of its activity as temperature higher than 60℃. The AjFT showed high structure stability in SDS as it present similar activity in 3% SDS. However, it lost half activity in 1M Guanidine-HCl or 3.5M Urea. To investigate the amino-acid residue around the active site, we have point mutate several positions of the recombinant AjFT and got 13 mutants. The preliminary data show that the activity of some mutant were decreased and some were increased. The roles of these residues were discussed.
關聯:	校內校外均不公開，學年度：97，80 頁
顯示於類別:	[生物科技系(所)] 博碩士論文

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