定点诱变G5-淀粉酶

Site-directed Mutagenesis of G5-amylase

利用定点突变技术，分别改变位于 Ｇ５淀粉酶氨基酸序列上高度保守区段内的３个氨基酸 Ｔｒｐ５７、 Ｔｙｒ１３９和 Ｌｙｓ１８８。其中， Ｔｒｐ５７和 Ｔｙｒ１３９分别被 Ｈｉｓ、 Ｐｈｅ、 Ｌｅｕ 和 Ｔｙｒ 取代， Ｌｙｓ１８８分别被 Ａｒｇ 和 Ｑｌｎ 取代，共得到９种单点突变基因。这些突变基因均在大肠杆菌中获得表达。对这些突变基因所表达的 Ｇ５淀粉酶的水解活性及其变化进行了初步分析。发现这些突变酶对淀粉和 Ｇ５的水解活性有不同程度的下降，而且对 Ｇ５的水解活性的下降尤为明显，其中，个别突变酶在基本保持对淀粉的水解活性的同时，对 Ｇ５的水解活性有大幅度的下降。

The residues in catalytic active site of G5 amylase were altered by site directed mutagenesis.Tryptophan 57 and Tyrosine 139 were replaced with histidine,leucine,phenylalanine and tyrosine;lysine 188 was replaced with arginine,glutamine.Nine single site mutant genes were obtained after Trp57,Tyr 139 and Lys188 were replaced.Hydrolysis activity of mutant G5 amylases to starch and G5 had been changed differently.They could all express in E.coli.The result indicated that the hydrolysis activity of some mutant G5 amylases to starch and G5 reduced differently,especially their hydrolysis activity to G5 reduced obviously.One or two among these mutant G5 amylases maintained constitutive hydrolysis activity to starch,but lost most of the hydrolysis activity to G5 at the same time.