利用定点突变分析海藻糖合酶的功能

Functional Analysis of Trehalose Synthase in Meiothermus ruber CBS-01 by Site-directed Mutation

我们通过对来自红色亚栖热菌(Meiothermus ruber)CBS-01中的海藻糖合酶(Trehalose synthase)序列比对及三维模型构建,我们构建了D200G/H165R,R227C,R392A三个定点突变体,检测其对麦芽糖及海藻糖的转化能力。结果发现:在50°C时,D200G/H165R、R392A基本失去其原有活性,而R227C产生海藻糖的能力降低。37°C时,D200G/H165R失去转化能力,而R392A及R227C保有部分能力。因此我们推测,R392位点可能是维持酶的结构及热稳定性的关键位点,而D200位点在反应过程中也起重要作用。

After constructed a 3D-Model and make the multiple sequence alignment of amino acid sequences of trehalose synthase from Meiothermus ruber CBS-01, we performed site-directed mutagenesis of D200G/H165R, R227C, R392A. And the ablity of convertion was detected. D200G/H165R and R392A lost their activities basically, while the ability of convertion of R227C declined at 50℃. When reacted at 37℃, D200G/H165R lost its activity, while R392A and R227C dropped their ability. At last, we found that R392 and D200 had important role on activity of enzyme, while R227 had little affection.