Journal
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Volume 116, Issue -, Pages 563-571Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.ijbiomac.2018.05.045
Keywords
Nitrilase; Homology modeling; Gene site saturation mutagenesis; Iminodiacetonitrile; Iminodiacetic acid
Funding
- National Natural Science Foundation of China [21476210]
- Natural Science Foundation of Zhejiang Province [R3110155, Z4090612]
- Public Welfare Technology Applied Research Projects of Zhejiang Province [2014C33223]
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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 V-max and k(cat), of Mut-F168V/T201N/S192F/M191T/F192S were enhanced 1.23 and 1.23-fold, while the K-m 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. (C) 2018 Elsevier B.V. All rights reserved.
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