表面电荷突变与胰蛋白酶底物专一性的改造

Alteration of Substrate Specificity of Trypsin by Surface Charge Mutation

分别利用酶切重组和“３＋１”引物ＰＣＲ定点突变的方法构建了三个胰蛋白酶表面电荷双突变体：Ｒ６２Ｄ＋Ｋ９７Ｅ、Ｒ６２Ｄ＋Ｋ１７５Ｅ和Ｋ９７Ｅ＋Ｋ１７５Ｅ．对三者在Ｅ．ｃｏｌｉＸ－９０菌中的表达产物进行了动力学测定，分别得到了三种双突变体在两种ｐＨ条件下，水解ＴＡＭＥ、ＴＬＭＥ两种底物的动力学数据．结果表明，Ｒ６２Ｄ＋Ｋ１７５Ｅ和Ｋ９７Ｅ＋Ｋ１７５Ｅ在ｐＨ６．８５时，对两种底物的催化活性与野生型相比下降了２～３个数量级，当ｐＨ升高至８．８５时，它们的活性基本丧失；双突变体Ｒ６２Ｄ＋Ｋ９７Ｅ虽然催化活性也有所降低，但随着ｐＨ的升高，它对Ｌｙｓ底物的特异性（选择性系数２５倍于Ａｒｇ底物）转变为对Ａｒｇ底物略高的特异性，基本符合分子设计．实验结果还表明，各种双突变体催化活性的降低主要是由于酶和底物的亲和力降低引起的．

Three trypsin double mutants R62D+K97E,R62D+K175E and K97E+K175E which have changed surface charges were constructed by recombination of restriction fragments and “3+1” primer PCR,a revised PCR site directed mutagenesis method.The three trypsin double mutants were expressed in E.coli X 90 strain with pT3 as the vector,and purified.Kinetic properties of the mutants were then determined under two pH and TAME,TLME substrates.The catalytic activities of R62D+K175E and K97E+K175E dropped 2～3 magnitudes at pH6 85,and they had no detectable activities at pH8 85.Though R62D+K97E appeared to have reduced catalytic activities at both pH,its preference toward Lys changed to Arg when the pH changed from 6 85 to 8 85.Analysis of kinetic properties also showed that the reduction of catalytic activities resulted mainly from the decreased affinity of the mutant enzymes for substrates.