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National Chung Hsing University Institutional Repository - NCHUIR > 農業暨自然資源學院 > 生物科技學研究所 > 依資料類型分類 > 碩博士論文 >  Bacillus subtilis 6-磷酸葡萄糖異構酵素中影響熱穩定性之主要胺基酸的探討

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/97939

標題: Bacillus subtilis 6-磷酸葡萄糖異構酵素中影響熱穩定性之主要胺基酸的探討
The amino acid residues affecting the thermostability of phosphoglucose isomerase from Bacillus subtilis
作者: 蘇致安
Contributors: 孟孟孝
關鍵字: 6-磷酸葡萄糖異構酵素;熱穩定;定點突變;活性半衰期
phosphoglucose isomerase;Bacillus subtilis;Bacillus stearothermophilus;thermounstability;site-directed mutagenesis
日期: 2003
Issue Date: 2012-09-04 09:24:02 (UTC+8)
Publisher: 生物科技學研究所
摘要: 6-磷酸葡萄糖異構酵素(phosphoglucose isomerase,EC;簡稱PGI)是一個普遍存在一般生物體內的酵素,其功能在催化六磷酸葡萄糖與六磷酸果糖之間的轉換。此酵素的基因已經從許多不同的有機體中被選殖出來。分離自嗜中溫性Bacillus subtilis的6-磷酸葡萄糖異構酵素(PgiBS)在胺基酸序列上與分離自嗜熱性Bacillus stearothermophilus的6-磷酸葡萄糖異構酵素-A(PgiA)有81﹪的相同性及91﹪的相似性。酵素PgiA在62℃時的活性半衰期約為86.8小時,然而酵素PgiBS在相同溫度下卻極不穩定,其活性半衰期為0.52分鐘。為了找出造成PgiBS熱不穩定之結構上的決定因素,我們試著利用定點突變的方法置換PgiBS蛋白質序列上與PgiA不同的胺基酸,並分析突變後對蛋白質熱穩定性的影響。我們針對PgiBS上數個胺基酸做突變,並送入大腸桿菌(E. coli.)中大量表現。其中有兩個突變蛋白質,I117V與M371L,相較於野生株蛋白質,在活性半衰期上分別有3.8及3.5倍的增加;然而,Y307F突變蛋白的活性半衰期卻降低了3.2倍。結合I117V與M371L,我們得到一個雙重突變蛋白,其活性半衰期提高了5.6倍。未來,我們期望得到一個包含最少胺基酸取代的突變蛋白,擁有與PgiA相當的耐熱性。這樣的突變蛋白能幫助我們找出影響PgiBS熱穩定的主要胺基酸。
The gene of phosphoglucose isomerase (PGI , EC, a ubiquitous house keeping enzyme catalyzing the inter-conversion between D-glucopyranose 6-phosphate and D-fructofuranose 6-phosphate, has been isolated from a variety of organisms. The enzyme isolated from mesophilic Bacillus subtilis (PgiBS) shares 81﹪identity and 91﹪similarity in amino acid sequence with that from thermophilic Bacillus stearothermophilus (PgiA). The half-life (t1/2) of PgiA is about 86.8 hours at 62℃; whereas, PgiBS is unstable at the same temperature with a t1/2 of 0.52 minutes. In order to find out the structural determinants resulting in the thermounstability of PgiBS, we attempted to replace each of the nonconsensus amino acids of PgiBS by site-directed mutagenesis, and analyze the mutational effects on protein thermostability. Several mutated proteins have been generated and expressed in E. coli. Two of them, I117V and M371L, have gained tolerance against thermoinactivation with a 3.8-fold and 3.5-fold increase in t1/2, respectively; whereas the mutant protein, Y307F, has become more vulnerable to heat with a decreasing t1/2 of 3.2-fold. Combining the two mutantions, I117->V and M371->L, the double mutant protein has an increasing t1/2 of 5.6-fold. In the future, we will continue to analyze the effects of other nonconsensus amino acids, and try to generate a mutated protein as stable as PgiA with fewest substitutions. This will help us to find out the key residues responsible for the thermostabilizing of PgiBS.
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