大肠杆菌CM/PDT抗反馈抑制突变体的构建及其性质

Construction and Characterization of E.coli CM/PDT Mutants with Feedback-inhibition Resistance

为了获得具有高催化活性且抗反馈抑制的大肠杆菌分支酸变位酶 预苯酸脱水酶 (chorismatemutase prephenatedehydrataseCM PDT) [EC5 .4 .99.5 EC4 .2 .1.5 1],通过相关菌种CM PDT氨基酸序列同源比较 ,寻找高度保守位点 .用定点突变及PCR法构建突变酶M1(缺失 30 4T、30 5G、Q30 6K)、M2 (缺失W 338)、M3(缺失 30 1～ 386位氨基酸 )、M32 9(E32 9A)和M374 (C374A) ,野生型及各突变型基因与pET2 8a(+ )载体连接后 ,表达融合蛋白 .在非变性条件下 ,由TALON金属螯合亲和层析柱纯化野生型和突变体的酶蛋白 .酶活性测定表明 ,突变体M3的PDT活性下降为野生型活性的 2 9% ,但保持了CM活性 .突变体M374保持了CM ,PDT两种酶的活性 ,突变体M1、M2、M32 9的CM ,PDT活性有一定程度的提高 .酶抗反馈抑制作用检测表明 ,突变体M3、M374解除了苯丙氨酸的反馈抑制作用 ,M1、M2、M32 9部分解除了苯丙氨酸的反馈抑制作用 .与含野生型pheA基因的E .coliBL2 1菌株相比 ,含突变基因的E .coliBL2 1菌株对 10mmol L的苯丙氨酸代谢类似物具有强的抗反馈抑制作用 ,其中M1,M2 ,M3对 2 0mmol

In order to obtain E.coli CM/PDT mutants with high catalytic activities and feedback-inhibition resistance, site-directed mutagenesis and PCR were used to construct five mutants on the basis of homology of the related amino acid sequences of CM/PDT: M1(T304, G305 deleted, Q306K), M2(W338 deleted), M3(301-386aa deleted), M329 (E329A) and M374(C374A). The five mutant genes were inserted into prokaryotic fusion-expression vector pET28a(+). The expressed products were purified by TALON affinity chromatography resin in native condition. The specific activities and feedback-inhibition resistance of the purified enzymes were determined. Catalytic activities of PDT of M3 retained only 29% of the wild type, but the catalytic activity of CM was retained. Bifunctional catalytic activities of M374 were retained and the bifunctional catalytic activities of mutants M1, M2, M329 were improved to different extent. Mutant M3 and M374 were of great feedback-resistance to phenylalanine, M1, M2 and M329 also showed feedback-resistance to some extent. E.coli BL21 harbouring mutant pheA were all resistant to 10 mmol/L phenylalanine analogue while the wild type was not. Furthermore, M1, M2, M3 were resistant to 20 mmol/L phenylalanine analogue.