定点突变Ca^(2+)平衡位点对粘质沙雷氏菌脂肪的影响

The Effect of Site-Directed Mutation about Ca^(2+) Binding Site on the Serratia marcescens Lipase lip A

为了研究粘质沙雷氏菌(Serratia marcescens)脂肪酶lip A中的Ca^(2+)对酶活力的影响,采用定点突变的方法对平衡Ca^(2+)的位点D388进行定点突变,对突变体进行诱导表达后,纯化目的蛋白并进行酶学性质分析。与野生酶相比,突变酶的最适温度由40℃降低到25℃,最适p H值由8.5降到8.0,T_(1/2)值比野生酶提高了2℃。由此可知,该Ca^(2+)对脂肪酶lip A酶学性质有着至关重要的作用。

In order to study the role of the Ca2＋ in li A of Serratia marcescen,site-directed mutation was done on the site of D388 which binds a Ca2＋.After inducing expression,characteristics of the mutant enzyme were analyzed using purified protein.Compared to the wild type enzyme,the optimum temperature of the mutant enzyme decreased significantly from 40℃ to 25℃and the optimum p H decreased from 8.5 to 8.0,and the value of T1/2 increased by 2℃.These results indicated that the Ca2＋which binded by D388 site might be crucial for the activity and characteristics of the enzyme.