Title
Significant improvement of the nitrilase activity by semi-rational protein engineering and its application in the production of iminodiacetic acid
Author
Zhi-Qiang Liu, Ming-Ming Lu, Xin-Hong Zhang, Feng Cheng, Jian-Miao Xu, Ya-Ping Xue, Li-Qun Jin, Yuan-Shan Wang, Yu-Guo Zheng
Year
2018
Journal
International Journal of Biological Macromolecules
Abstract
Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacturing of chelating agents, glyphosate herbicides and surfactants. To improve activity and tolerance to the substrate for IDA production, Acidovorax facilis nitrilase was selected for further modification by the gene site saturation mutagenesis method. After screened by a two-step screening method, the best mutant (Mut-F168V/T201N/S192F/M191T/F192S) was selected. Compared to the wild-typenitrilase, Mut-F168V/T201N/S192F/M191T/F192S showed 136% improvement in specific activity. Co2+ stimulated nitrilase activity, whereas Cu2+, Zn2+ and Tween 80 showed a strong inhibitory effect. The Vmax and kcat of Mut-F168V/T201N/S192F/M191T/F192S were enhanced 1.23 and 1.23-fold, while the Kmwas decreased 1.53-fold. The yield of Mut-F168V/T201N/S192F/M191T/F192S with 453.2 mM of IDA reached 71.9% in 5 h when 630 mM iminodiacetonitrile was used as substrate. This study indicated that mutant nitrilase obtained in this study is promising in applications for the upscale production of IDAN.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Protein folding, Thermal stability, Biochemistry