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

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

doi:10.1016/j.ijbiomac.2018.05.045

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

Int J Biol Macromol. 2018 Sep:116:563-571. doi: 10.1016/j.ijbiomac.2018.05.045. Epub 2018 May 10.

Authors

Zhi-Qiang Liu¹, Ming-Ming Lu¹, Xin-Hong Zhang², Feng Cheng¹, Jian-Miao Xu¹, Ya-Ping Xue¹, Li-Qun Jin¹, Yuan-Shan Wang¹, Yu-Guo Zheng³

Affiliations

¹ Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China.
² Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China; Department of Biological and Environmental Engineering, Hefei University, Hefei 230601, China.
³ Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China. Electronic address: zhengyg@zjut.edu.cn.

PMID: 29753012
DOI: 10.1016/j.ijbiomac.2018.05.045

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 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.

Keywords: Gene site saturation mutagenesis; Homology modeling; Iminodiacetic acid; Iminodiacetonitrile; Nitrilase.

MeSH terms

Amino Acid Substitution*
Aminohydrolases* / chemistry
Aminohydrolases* / genetics
Bacterial Proteins* / chemistry
Bacterial Proteins* / genetics
Comamonadaceae* / enzymology
Comamonadaceae* / genetics
Mutagenesis, Site-Directed*
Recombinant Proteins / chemistry

Substances

Bacterial Proteins
Recombinant Proteins
Aminohydrolases
nitrilase