大肠杆菌
突变体
异源表达
发酵
酶
生物化学
链霉菌
拉伤
异源的
化学
蛋白质工程
野生型
定向进化
辅因子
代谢工程
酶分析
生物
重组DNA
基因
细菌
遗传学
解剖
作者
Meng Shi,Demin Kong,Hui Zhang,Deming Rao,Tianlong Zhao,Jing Yang,Zhanzhi Liu,Sheng Chen,Fengshan Zhang,Jing Wu,Lei Wang
标识
DOI:10.1016/j.ijbiomac.2023.127995
摘要
Latex clearing protein from Streptomyces sp. strain K30 (LcpK30) is a natural oxidoreductase that can catalyse the cleavage of rubber through dioxygenation. It has significant potential applications in polymer degradation. However, its limited expression in engineered strains restricts its utility. This study aimed to enhance the soluble expression and enzyme activity of LcpK30 in E. coli BL21 (DE3) by optimizing fermentation conditions and making molecular modifications. The enzyme activity reached 5.05 U·mL−1 by optimizing the induction conditions, adding cofactors, and using chemical chaperones, which was 237.1 % of the initial case. Further enhancements in soluble expression were achieved through site mutations guided by the PROSS server, resulting in 8 out of 13 mutants with increased protein expression, a high positive mutation rate of 61.5 %. Subsequently, combined mutants were created by merging single mutants with enhanced protein expression and enzyme activity. The top three double mutants, G91D/S149A, G91D/A210H, and G91D/H296P, displayed expression levels at 173.3 %, 173.3 %, and 153.3 % of the wild-type LcpK30, respectively. These mutants also exhibited enhanced fermentation enzyme activity, reaching 149.5 %, 250.0 %, and 420.2 % compared to the wild-type, along with improved specific activities. This study provides insights for the efficient production of LcpK30 and a practical foundation for its application.
科研通智能强力驱动
Strongly Powered by AbleSci AI