枯草蛋白酶E的定点突变及其对酶性质的影响

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

用定点突变的方法研究S2 2 1C/P2 2 5A ,N118S/S2 2 1C/P2 2 5A ,D6 0N/S2 2 1C/P2 2 5A和Q10 3R/S2 2 1C/P2 2 5A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 :S2 2 1C/P2 2 5A突变使蛋白酶活性比枯草蛋白酶E低 730 0 0多倍 ,酯酶活性与蛋白酶活性之比是Subtiligase的 3倍 ;N118S/S2 2 1C/P2 2 5A突变使蛋白酶活性和酯酶活性分别比S2 2 1C/P2 2 5A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 ;D6 0N/N118S/S2 2 1C/P2 2 5A突变使蛋白酶活性比N118S/S2 2 1C/P2 2 5A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S2 2 1C/P2 2 5A突变体和Subtiligase的 3 3倍和 10 3倍 ;但是 ,Q10 3R/N118S/S2 2 1C/P2 2 5A突变使蛋白酶活性比N118S/S2 2 1C/P2 2 5A突变体增加 5倍 ,酯酶活性下降5 5倍 ,酯酶与蛋白酶活性之比下降 10 0 0倍。

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.