Chemostat selection of an Escherichia coli mutant containing permease with enhanced lactose affinity.
Chemostat selection of an Escherichia coli mutant containing permease with enhanced lactose affinity.
复制标题
含有乳糖亲和力增强的通透酶的大肠杆菌突变体的恒化器选择。
DOI：
10.1006/bbrc.1996.1032
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Wilson,TH
中科院分区：
文献类型：
--
作者：
Tsen,SD;Lai,SC;Pang,CP;Lee,JI;Wilson,TH
使用提供有限乳糖的趋化器来询问是否有可能产生和分离任何允许细胞生长更快的大肠杆菌乳糖渗透酶突变体。恒化培养物的渗透酶和β-半乳糖苷酶活性最初一起上升，达到一个平稳期。30天后，前者单独进行第二次增加。从这个培养中，分离出一个生长更快的突变体。对其渗透酶基因进行克隆、测序，发现有一个碱基对发生了变化。199位的胸腺嘧啶变成了鸟嘌呤，导致67号丝氨酸被丙氨酸取代。质粒中携带该突变体的细胞在10 μM乳糖环境中比亲本生长更快。突变体对乳糖的渗透酶的kmf为1.4 mM，约为野生型的一半。因此，可以从趋化调控中选择对底物有较高亲和力的突变体。
Chemostats supplied with limited lactose were used to ask whether it was possible to generate and isolate any mutant ofEscherichia colilactose permease which allowed cells to grow faster. The permease and β-galactosidase activities of the chemostat culture initially rose together to reach a plateau. After 30 days, the former underwent a second increase alone. From this culture, a faster-growing mutant was isolated. Its permease gene was cloned, sequenced, and found to have a single base pair changed. Thymine at position 199 was changed to guanine, resulting in serine 67 being substituted by alanine. Cells bearing this mutant in the plasmid could grow faster than parents in 10 μM lactose. The Kmof the mutant permease toward lactose was 1.4 mM, about half of the wild-type value. Thus, a mutant with higher affinity for substrate could be selected from the chemostat.