- 學位論文
含澱粉酶菌( Exiguobacterium sp. )的分離、鑑定與特性分析
Isolation and Characterization of α-Amylase Containing Bacteria (Exiguobacterium sp.)
摘要
α-澱粉酶家族擁有三項共同特徵：在α-糖甘鍵上作用、擁有包含催化端的花籃狀蛋白結構、基因上有4個高度保留區分別和形成催化端與穩定花籃狀蛋白構形有關。本實驗利用基因轉殖方法，嘗試轉殖α-澱粉酶基因片段，透過隨機水解細菌染色體、轉殖入宿主細胞，製做Exiguobacterium sp.的基因圖譜，並以活性染色方式篩選菌株。成功轉殖的片段再經過修飾後轉殖入表達載體，配和表達宿主經過1 mM IPTG誘導後，透過蛋白質膠體染色可看出酵素明顯活性增加。 原菌酵素活性測試為48.82 U/mg，酵素最適pH為6，最適反應溫度為37℃。
並列摘要
The α-amylase family has three common features：Act on α-glycosidic bonds and hydrolyze the bond to α-anomeric oligosacchrides or forming α-1,4 or α-1,6 bond、they all have the (β/α)8 or TIM barrel protein structure contain the catalytic site、all of them have four highly conserved regions in their primary sequence which contain the amino acids that form the catalytic site and TIM barrel. DNA library was made by using partial digestion to digest Exiguobacterium sp. chromosome.Then clone into host cell selected by activity staining. Modified the sequence and clone again into pET expression vector system. Add 1 mM IPTG for induce a large amount of protein. Primary supernatant of Exiguobacterium sp. was concentrate and test by DNSA. The special activity was 48.82 U/mg. While the condense supernatant has the optimal condition on pH 6 and 37℃.