摘要
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.
基金
国家863计划基金
关键词
胰蛋白酶突变体
底物专一性
表面电荷
Mutant trypsin,Substrate specificity,pH dependence,Surface charge