枯草蛋白酶E的突变体

Mutants of Subtilisin E

Ser236位于横贯枯草蛋白酶E的α螺旋末端,远离催化活性中心,Ser236的突变不会对酶的活性产生大的影响。用定点突变的方法对枯草蛋白酶E的基因进行改造引入Ser236Cys,可能会形成分子间二硫键,有利于提高酶的稳定性。Ser236Cys变体酶(BP1)活性是野生型蛋白酶E的15倍,热稳定性提高3倍;进一步在其他位点引入突变的变体酶BU1(A1a15Asp/Gly20His/Ser236Cys)和BW1(Ser24His/Lys27Asp/Ser236Cys)活性都比野生型蛋白酶E低,但BW1的稳定性稍高于野生型蛋白酶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.