Engineering the residues on "A" surface and C-terminal region to improve thermostability of nitrilase

Zhe Xu; Ting Cai; Neng Xiong; Shu-Ping Zou; Ya-Ping Xue; Yu-Guo Zheng

doi:10.1016/j.enzmictec.2018.03.001

Engineering the residues on "A" surface and C-terminal region to improve thermostability of nitrilase

Enzyme Microb Technol. 2018 Jun:113:52-58. doi: 10.1016/j.enzmictec.2018.03.001. Epub 2018 Mar 5.

Authors

Zhe Xu¹, Ting Cai¹, Neng Xiong¹, Shu-Ping Zou¹, Ya-Ping Xue², Yu-Guo Zheng¹

Affiliations

¹ Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, 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; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, China. Electronic address: xyp@zjut.edu.cn.

PMID: 29602387
DOI: 10.1016/j.enzmictec.2018.03.001

Abstract

Nitrilases can hydrolyze nitriles to corresponding carboxylic acids in one single step, which have great potential as valuable biocatalysts for chemical synthesis. However, the poor thermostability of the nitrilases restrict their applications in industry. In this work, error-prone PCR and site-directed mutagenesis were utilized to improve the thermostability of nitrilases. Several mutants (AcN-Q339K, AcN-Q343K, AcN-T201F, AcN-T201W, AcN-T201L, AcN-T201I) were obtained with dramatically improved thermostability. The best mutant AcN-T201F/Q339K/Q343K exhibited about 14-fold longer half-life at 45 °C. The result of homology modeling suggested that the site 201, which was located on the "A" surface (the dimer interaction), played an important role in the oligomerization of nitrilase and the stabilization of substrate binding pocket. The Phe substitution on site 201 was selected in protein engineering of nitrilase LNIT5, which also demonstrated an improvement of thermostability. In addition, lysine substitution on Q339 and Q343 which brought positive charges to the α helix in the C-terminal region stabilized the surface.

Keywords: Directed evolution; Gabapentin; Nitrilase; Protein engineering; Thermostability.

MeSH terms

Aminohydrolases / chemistry*
Aminohydrolases / genetics
Aminohydrolases / metabolism
Enzyme Stability
Escherichia coli / enzymology*
Escherichia coli / genetics
Escherichia coli / metabolism
Models, Molecular
Mutagenesis, Site-Directed
Mutation
Nitriles / chemistry
Nitriles / metabolism*
Protein Conformation
Protein Engineering / methods*
Temperature
Thermodynamics

Substances

Nitriles
Aminohydrolases
nitrilase