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

吉林大学学报(理学版) ›› 2019, Vol. 57 ›› Issue (5): 1267-1274.

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

高云娜, 韩彩静, 詹冬玲, 方丽, 闵伟红

吉林农业大学食品科学与工程学院, 长春 130118

收稿日期:2018-12-04 出版日期:2019-09-26 发布日期:2019-09-20
通讯作者: 闵伟红 E-mail:minwh2000@vip.163.com

Construction and Enzymatic Properties of Double Mutant M372I-T379S of Aspartokinase from Corynebacterium pekinense#br#

GAO Yunna, HAN Caijing, ZHAN Dongling, FANG Li, MIN Weihong

College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China

Received:2018-12-04 Online:2019-09-26 Published:2019-09-20
Contact: MIN Weihong E-mail:minwh2000@vip.163.com

摘要/Abstract

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-T379S AK与WTAK相比, V_max分别提高了13.77,15.02,15.60倍, K_m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,2.3 h； M372I-T379S AK最适温度为30 ℃, 比WT AK高2 ℃；当浓度为1~10 mmol/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.

关键词: 天冬氨酸激酶, 定点突变, 酶动力学, 酶学性质

Abstract: In order to construct mutants with high enzyme activity of aspartokinase (AK) and weaken or relieve the feedback inhibition of Lys (lysine), mutants M372I,T379S and M372I-T379S 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 V_max of mutants M372I,T379S and M372I-T379S AK increases 13.77,15.02 and 15.60 times, respectively, and the values of K_m and n are lower than that of WTAK, the optimum pH values of mutants M372I,T379S and M372I-T379S 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 and 2.3 h, respectively. The optimal temperature of M372I-T379S AK 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.

Key words: aspartokinase, site\, directed mutagenesis, enzymatic kinetics, enzymatic property

中图分类号:

高云娜, 韩彩静, 詹冬玲, 方丽, 闵伟红. 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[J]. 吉林大学学报(理学版), 2019, 57(5): 1267-1274.

GAO Yunna, HAN Caijing, ZHAN Dongling, FANG Li, MIN Weihong. Construction and Enzymatic Properties of Double Mutant M372I-T379S of Aspartokinase from Corynebacterium pekinense#br#[J]. Journal of Jilin University Science Edition, 2019, 57(5): 1267-1274.

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