Significant improvement of the nitrilase activity by semi-rational protein engineering and its application in the production of iminodiacetic acid

International Journal of Biological Macromolecules
Zhi-Qiang LiuYu-Guo Zheng

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-type nitrilase, 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 Km was 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.

Citations

Nov 26, 2019·Indian Journal of Microbiology·Pratyoosh Shukla
Jul 18, 2018·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Feng-Jie CuiDi Wu
Oct 14, 2020·Critical Reviews in Biotechnology·Ji-Dong ShenYu-Guo Zheng
Mar 12, 2020·Biotechnology and Bioengineering·Misha AliQayyum Husain

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