Engineering Nitrilase with Improved Enantioselectivity for Specific Hydrolysis and Hydration Reactions
Xiao-Ling Tang1,2, Ye-Tao Zhang1,2, Xiao-Xiao Liu1,2
1College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, P. R. China.
Journal of Agricultural and Food Chemistry
|December 26, 2025
Summary
Protein engineering of rice nitrilase (OsNIT) enhanced enantioselectivity for producing chiral amides and acids. This study offers a strategy to control enzyme properties for valuable chemical synthesis.
Area of Science:
- Biocatalysis
- Protein Engineering
- Enzyme Technology
Background:
- Nitrilases are biocatalysts converting nitriles to carboxylic acids and ammonia.
- Recent studies highlight nitrilases' hydration activity for amide generation.
- Enantioselectivity is crucial for nitrilase applications in chiral synthesis.
Purpose of the Study:
- To engineer the nitrilase from *Oryza sativa* (*Os*NIT) for enhanced enantioselectivity.
- To develop novel enzymes with dual regulation of specificity and enantioselectivity.
- To produce high-purity chiral carboxylic acids or amides.
Main Methods:
- Protein engineering of *Os*NIT.
- Mutagenesis to enhance hydration and hydrolysis enantioselectivity.
- Substrate: racemic mandelonitrile.
Main Results:
- An enhanced enantioselective hydration mutant produced (R)-mandelamide with 91.40% enantiomeric excess (e.e.).
- An improved enantioselective hydrolysis mutant synthesized (R)-mandelic acid with 96.45% e.e.
- Structural-functional analysis provided insights into altered catalytic performance.
Conclusions:
- Protein engineering can significantly enhance nitrilase enantioselectivity for specific reactions.
- The study provides a valuable strategy for regulating multiple catalytic properties of nitrilases.
- Engineered nitrilases show promise for efficient production of high-purity chiral compounds.
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