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An-Di Dai ◽  
Xiao-Ling Tang ◽  
Zhe-Ming Wu ◽  
Jiang-Tao Tang ◽  
Ren-Chao Zheng ◽  

Nitrilase-catalyzed hydrolysis of 2-chloronicotinonitrile (2-CN) is a promising approach for efficient synthesis of 2-chloronicotinic acid (2-CA). Development of nitrilase with ideal catalytic properties is crucial for the biosynthetic route with industrial potentail. Herein, a nitrilase from Rhodococcus zopfii ( Rz NIT), which showed much higher hydration activity than hydrolysis activity, was designed for efficient hydrolysis of 2-CN. Two residues (N165 and W167) significantly affecting the reaction specificity were precisely identified. By tuning these two residues, a single mutation of W167G with abolished hydration activity and 20-fold improved hydrolysis activity was obtained. Molecular dynamics simulation and molecular docking revealed that the mutation generated a larger binding pocket, causing the substrate 2-CN bound more deeply in the pocket and the formation of delocalized π bond between the residues W190 and Y196, which reduced the negative influence of steric hindrance and electron effect caused by chlorine substituent. With mutant W167G as biocatalyst, 100 mM 2-CN was exclusively converted into 2-CA within 16 h. The study provides useful guidance in nitrilase engineering for simultaneous improvement of reaction specificity and catalytic activity, which are highly desirable in value-added carboxylic acids production from nitriles hydrolysis. Importance 2-CA is an important building block for agrochemicals and pharmaceuticals with rapid increase in demand in recent years. It is currently manufactured from 3-cyanopyridine by chemical methods. However, during the final step of 2-CN hydrolysis under high temperature and strong alkaline conditions, by-product 2-CM was generated except for the target product, leading to low yield and tedious separation steps. Nitrilase-mediated hydrolysis is regarded as a promising alternative for 2-CA production, which proceeds under mild conditions. Nevertheless, nitrilase capable of efficient hydrolysis of 2-CN was not reported till now, since the enzymes showed either extremely low activity or surprisingly high hydration activity towards 2-CN. Herein, the reaction specificity of Rz NIT was precisely tuned through a single site mutation. The mutant exhibited remarkably enhanced hydrolysis activity without formation of by-products, providing a robust biocatalyst for 2-CA biosynthesis with industrial potential.

Izabel C. Medeiros-Costa ◽  
Eddy Dib ◽  
Florent Dubray ◽  
Simona Moldovan ◽  
Jean-Pierre Gilson ◽  

ACS Catalysis ◽  
2022 ◽  
pp. 1247-1257
Jiaqi Li ◽  
Anusheela Das ◽  
Qing Ma ◽  
Michael J. Bedzyk ◽  
Yosi Kratish ◽  

Henrique Rasia Bosi ◽  
Marcelo Costamilan Rombaldi ◽  
Thamyres Zaniratti ◽  
Fernanda Oliveira Castilhos ◽  
Mariana Sbaraini ◽  

2021 ◽  
Vol 7 (2) ◽  
Vromans Anna Maria ◽  
Williams Josiah ◽  
Bashyam Arjun M ◽  
Huang William W ◽  
Feldman Steven R ◽  

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