枯草蛋白酶E的定点突变及其对酶性质的影响

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73000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1000倍。

Site-directed Mutagenesis and Effects on the Enzymatic Properties of Subtilisin E

Site\|directed mutagenesis was used to investigate the effects of S221C/P225A,N118S/S221C/P225A,D60N/N118S/S221C/P225A and Q103R/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.