热稳定性
突变体
酶
酶动力学
定向进化
异构酶
木糖
生物化学
化学
生物
基因
活动站点
发酵
作者
Yun Fei Feng,Zhongji Pu,Zhu Li,Mianbin Wu,Lirong Yang,Haoran Yu,Jianping Lin
标识
DOI:10.1016/j.enzmictec.2022.110054
摘要
D-Allulose 3-epimerase (DAE) is promising to be used for the production of the rare sugar D-allulose in industry. However, the poor thermostability and low catalytic efficiency limited its large-scale industrial applications. A dual-enzyme screening method was developed to measure the activity of the D-allulose 3-epimerase from Clostridium cellulolyticum H10 by employing a xylose isomerase, enabling high-throughput screening of mutants with higher thermostability. After two rounds of directed evolution, the H56R, Q277R, H56R/Q277R and H56R/Q277R/S293R variants were obtained with 1.9, 1.8, 3.5 and 7.1 °C improvement in T505, the temperature at which the enzyme activity becomes half of the original after the 5 min treatment and 3.1-, 4.2-, 4.4- and 9.47- fold improvement in the half life at 60 °C, respectively, compared with the wild-type enzyme. Among them, triple mutant H56R/Q277R/S293R showed significant improvement in kcat/Km compared to the wild type enzyme. Molecular dynamics simulations provided the insights into improving the thermostability by three arginine mutations. The research will aid the development of industrial biocatalysts for the production of D‑allulose.
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