Abstract
Purpose
We screened nitrilases with significant nitrile hydratase activity to exploit their potential in benzylic amide biosynthesis. We also investigated the factors affecting their hydration activity to support further research on benzylic amide production by nitrilase.
Methods
A sequence-based screening method using previously reported crucial positions identified to be essential for amide-forming capacity of nitrilase (referred to as “amide-formation hotspots”) as molecular probes to identify putative amide-forming nitrilases.
Results
Based on the previously reported “amide-formation hotspots,” we identified a nitrilase NitPG from Paraburkholderia graminis DSM 17151 that could produce a significant amount of mandelamide toward mandelonitrile and exhibited general hydration activity toward various benzylic nitriles. The time-course experiment with NitPG demonstrated that amide was also a true reaction product of nitrilase, suggesting that the nitrile catalysis by amide-forming nitrilase could be a post-transition state bifurcation-mediated enzymatic reaction. Further research demonstrated that low temperature, metal ion addition, and specific substrate structure could profoundly improve the amide formation capability of nitrilase.
Conclusions
NitPG with broad hydration activity is a potential candidate for the enzymatic synthesis of benzylic amides for biotechnological applications. Studying the effect of nitrilase hydration activity could promote our understanding of the factors that influence amide and acid distribution.
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Funding
This work is supported by the grant from the National Key Research and Development Program of China (Grant No. 2021YFC2102100).
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KZ and TZP: conceived and designed the study. KZ: conducted the literature search and performed the experiments. KZ and LZW: were involved in the analysis and interpretation of data. KZ: drafted the manuscript. HLW, YHR and DZW: The study was supervised and tutored. All authors read and approved the final manuscript.
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Zhang, K., Pan, T., Wang, L. et al. Screening and characterization of a nitrilase with significant nitrile hydratase activity. Biotechnol Lett 44, 1163–1173 (2022). https://doi.org/10.1007/s10529-022-03291-6
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DOI: https://doi.org/10.1007/s10529-022-03291-6