Improvement of multicatalytic properties of nitrilase from Paraburkholderia graminis for efficient biosynthesis of 2‐chloronicotinic acid

Abstract

Nitrilases are promising biocatalysts to produce high‐value‐added carboxylic acids through hydrolysis of nitriles. However, since the enzymes always show low activity and sometimes with poor reaction specificity toward 2‐chloronicotinonitrile (2‐CN), very few robust nitrilases have been reported for efficient production of 2‐chloronicotinic acid (2‐CA) from 2‐CN. Herein, a nitrilase from Paraburkholderia graminis (PgNIT) was engineered to improve its catalytic properties. We identified the beneficial residues via computational analysis and constructed the mutant library. The positive mutants were obtained and the activity of the “best” mutant F164G/I130L/N167Y/A55S/Q260C/T133I/R199Q toward 2‐CN was increased from 0.14 × 10−3 to 4.22 U/mg. Its reaction specificity was improved with elimination of hydration activity. Molecular docking and molecular dynamics simulation revealed that the conformational flexibility, the nucleophilic attack distance, as well as the interaction forces between the enzyme and substrate were the main reason alternating the catalytic properties of PgNIT. With the best mutant as biocatalyst, 150 g/L 2‐CN was completely converted, resulting in 2‐CA accumulated to 169.7 g/L. When the substrate concentration was increased to 200 g/L, 203.1 g/L 2‐CA was obtained with yield of 85.7%. The results laid the foundation for industrial production of 2‐CA with the nitrilase‐catalyzed route.

Structure-Oriented Engineering of Amidase: Modification of Twisted Access Tunnel for Efficient Synthesis of 2-Chloronicotinic Acid

“…2-Chloronicotinic acid (2-CA) is an important and valuable building block for agrochemicals and pharmaceuticals, such as the herbicides diflufenican and nicosulfuron. , Industrial synthesis of 2-CA is mainly achieved by chemical methods; however, there are drawbacks including drastic reaction conditions with toxic reagents and low product purity and yields. , Therefore, enzymatic biosynthesis of 2-CA from 2-chloroniconitrile via either nitrilases or coupling of nitrile hydratase and amidase is regarded as a promising route. Nevertheless, the reported nitrilases showed low enzyme activity and surprising catalytic promiscuity .…”

Bidirectional Regulation of Nitrilase Reaction Specificity by Tuning the Characteristic Distances between Key Residues and Substrate

“…Biosynthesis of amides from the corresponding nitrile has attracted a great deal attention. Although nitrile hydratase has been studied as a biocatalyst to produce amides for a long time, it suffered from drawbacks, including low stereoselectivity, poor thermal stability, and being easily inhibited by α-amino or α-hydroxyl nitriles . Switching of nitrilase with specific hydration activity is promising to solve the bottlenecks of nitrile hydratase.…”

“…Although nitrile hydratase has been studied as a biocatalyst to produce amides for a long time, it suffered from drawbacks, including low stereoselectivity, poor thermal stability, and being easily inhibited by α-amino or α-hydroxyl nitriles. 11 Switching of nitrilase with specific hydration activity is promising to solve the bottlenecks of nitrile hydratase. Therefore, bidirectional regulation of the reaction specificity of nitrilases is of great significance to broaden the application scope of this kind of enzyme.…”