枯草蛋白酶E的定点突变及其对酶性质的影响

摘要

Site-directed mutagenesis was used to investigate the effects of S221C/P225A,N118S/S221C/P225A,D60N/ N118S/S221C/P225A and QI03R/N118S/S221C/P225A mutations on the properties of Subtilisin E. It was found that S221C/P225A mutant is 73 000-fold decreased in amidase activity than subtilisin E and 3-fold increased than subtiligase in the ratio of esterase/amidase; N118S/S221C/P225A mutant has 3.6-fold and 15-fold decreased in amidase and esterase activity respectively and as a result,it has a 4-fold lower in the ratio of amidase/esterase than S221C/P225A mutant;Although it has no effect on the esterase activity, D60N/N118S/S221C/P225A mutant enhanced its ratio of amidase/esterase by 15 fold,3.3-fold and 10.3 fold compared to N118S/S221C/P225A mutant,S221C/P225A mutant and subtiligase respectively; Q103R/N118S/S221C/P225A mutant,however, has a 5-fold enhanced in the amidase activity and 55-fold and 1000-fold decrease in the esterase activity and the ratio of esterase/amidase compared to N118S/S221C/P225A.%用定点突变的方法研究S221C／P225A，N118S／S221C／P225A，D60N／S221C／P225A和Q103R／S221C／P225A突变对蛋白酶活性，酯酶活性与蛋白酶活性之比的影响。结果表明：S221C／P225A突变使蛋白酶活性比枯草蛋白酶E低73 000多倍，酯酶活性与蛋白酶活性之比是Subtiligase的3倍；N118S／S221C／P225A突变使蛋白酶活性和酯酶活性分别比S221C／P225A突变下降3.6倍和15倍，酯酶与蛋白酶活性之比下降4倍，同时增加变体酶的热稳定性；D60N／N118S／S221C／P225A突变使蛋白酶活性比N118S／S221C／P225A突变体下降15倍，但对酯酶活性几乎没有影响，酯酶与蛋白酶活性之比增加14倍，分别是S221C／P225A突变体和Subtiligase的3.3倍和10.3倍；但是，Q103R／N118S／S221C／P225A突变使蛋白酶活性比N118S／S221C／P225A突变体增加5倍，酯酶活性下降55倍，酯酶与蛋白酶活性之比下降1 000倍。