北京棒杆菌天冬氨酸激酶M372I-T379S双突变体的构建及其酶学性质  

Construction and Enzymatic Properties of Double Mutant M372I-T379S of Aspartokinase from Corynebacterium pekinense

作　　者：高云娜 韩彩静[1] 詹冬玲[1] 方丽[1] 闵伟红[1] GAO Yunna;HAN Caijing;ZHAN Dongling;FANG Li;MIN Weihong(College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China)

机构地区：[1]吉林农业大学食品科学与工程学院

出　　处：《吉林大学学报（理学版）》2019年第5期1267-1274,共8页Journal of Jilin University:Science Edition

基　　金：国家自然科学基金(批准号:31771957);长春市科技创新“双十工程”项目(批准号:17SS030)

摘　　要：为构建高酶活力天冬氨酸激酶(aspartokinase,AK),并削弱或解除Lys(lysine)反馈抑制作用突变体,通过定点突变和高通量筛选技术构建突变体M372I,T379S和M372I-T379S,对野生型(WT)和突变体分别进行诱导表达、纯化及酶学性质表征.结果表明:突变体M372I,T379S和M372I-T379SAK与WTAK相比,Vmax分别提高了13.77,15.02,15.60倍,Km和n值均降低;最适pH值分别升高为8.0,8.5,8.5,且半衰期分别延长了1.0,0.9,2.3h;M372I-T379SAK最适温度为30℃,比WT AK高2℃;当浓度为1~10mmol/L时,突变体均削弱或部分解除了抑制剂Lys的反馈抑制作用.In order to construct mutants with high enzyme activity of aspartokinase(AK)and weaken or relieve the feedback inhibition of Lys(lysine),mutants M372 I,T379 Sand M372 I-T379 S were constructed by site-directed mutagenesis and high-throughput screening techniques.The wild type(WT)and mutants were induced,expressed and characterized by enzymatic properties.The results show that compared with the WTAK,the Vmaxof mutants M372 I,T379 Sand M372 I-T379 S AK increases 13.77,15.02 and 15.60 times,respectively,and the values of Kmand nare lower than that of WTAK,the optimum pH values of mutants M372 I,T379 Sand M372 I-T379 S AK are increased slightly,which are 8.0,8.5,8.5,and the half-life period of mutants are prolonged by 1.0,0.9 and2.3 h,respectively.The optimal temperature of M372 I-T379 SAK is 30℃,which is higher than WT AK 2 ℃.When the concentration is 1-10 mmol/L,the mutants weaken or partially relieve the feedback inhibition of Lys inhibitor.

关 键 词：天冬氨酸激酶 定点突变 酶动力学 酶学性质 

分 类 号：Q78[生物学—分子生物学]