Conversion of aliphatic 2-acetoxynitriles by nitrile-hydrolysing bacteria

Area of Science:

• Biocatalysis and Enzyme Engineering
• Organic Chemistry
• Microbial Biotechnology

Background:

• Selective hydrolysis of nitriles in the presence of ester groups is challenging.
• 2-acetoxynitriles are versatile substrates with both nitrile and ester functionalities.
• Bacterial nitrilases and nitrile hydratases are known to process nitrile groups.

Purpose of the Study:

• To investigate the enzymatic hydrolysis of 2-acetoxynitriles to obtain selective nitrile-hydrolyzing catalysts.
• To identify microorganisms and enzymes capable of chemoselectively hydrolyzing the nitrile group of 2-acetoxynitriles.
• To overcome side reactions and achieve efficient conversion to 2-acetoxycarboxylic acids.

Main Methods:

• Biotransformation of 2-acetoxynitriles (ABN, AHN, AFN, ATMB) using various bacterial strains.
• Analysis of metabolic products, including cyanide and aldehydes.
• Heterologous expression of nitrilase genes in recombinant Escherichia coli.
• Purification and characterization of enzymes.

Main Results:

• Wild-type strains produced cyanide and aldehydes, indicating esterase activity and spontaneous decomposition of intermediates.
• Sterically hindered substrate ATMB was not metabolized.
• Recombinant E. coli strains expressing nitrilases showed improved conversion.
• High overexpression of a pseudomonad nitrilase in E. coli achieved near-stoichiometric conversion of 2-acetoxynitriles to 2-acetoxycarboxylic acids.

Conclusions:

• Nitrilase enzymes are key to achieving chemoselective hydrolysis of nitriles in 2-acetoxynitriles.
• Recombinant E. coli overexpressing specific nitrilases offers an efficient biocatalytic route.
• This approach suppresses unwanted esterase activity and decomposition pathways, enabling targeted synthesis of 2-acetoxycarboxylic acids.