Nitrilase enzymes (nitrile aminohydrolase; ) catalyse the hydrolysis of nitriles to carboxylic acids and ammonia, without the formation of "free" amide intermediates. Nitrilases are involved in natural product biosynthesis and post translational modifications in plants, animals, fungi and certain prokaryotes. Nitrilases can also be used as catalysts in preparative organic chemistry. Among others, nitrilases have been used for the resolution of racemic mixtures. Nitrilase should not be confused with nitrile hydratase (nitrile hydro-lyase; ) which hydrolyses nitriles to amides. Nitrile hydratases are almost invariably co-expressed with an amidase, which converts the amide to the carboxylic acid. Consequently, it can sometimes be difficult to distinguish nitrilase activity from nitrile hydratase plus amidase activity. Mechanism Nitrilase was first discovered in the early 1960s for its ability to catalyze the hydration of a nitrile to a carboxylic acid. Although it was known at the time that nitrilase could operate with wide substrate specificity in producing the corresponding acid, later studies reported the first NHase (nitrile hydratase) activity exhibited by nitrilase. That is, amide compounds could also be formed via nitrile hydrolysis. Further research has revealed several conditions that promote amide formation, which are outlined below. Early release of the enzyme-bound substrate after the first water hydrolysis followed by delayed addition of the second water Low temperature and increased pH conditions. For bioconversions by nitrilase for most bacteria and fungi, the optimal pH range is between 7.0-8.0 and the optimal temperature range is between 30-50℃. Electron withdrawing groups at the ⍺-position Below is a list of steps involved in transforming a generic nitrile compound with nitrilase: The electrophilic carbon of the nitrile is subject to nucleophilic attack by one of the two SH groups on nitrilase. The thioimidate formed is subsequently hydrolyzed to the acylenzyme and ammonia is created as a byproduct. The acylenzyme can undergo one of two pathways depending on the conditions highlighted above: Further hydrolyzation of the acylenzyme with water produces the carboxylic acid and the regenerated enzyme. The acylenzyme is hydrolyzed by ammonia, displacing the enzyme and forming the amide product. Structure Most nitrilases are made up of a single polypeptide ranging from 32-45 kDa, and its structure is an ⍺-β-β-⍺ fold. The favored form of the enzyme is a large filament consisting of 6-26 subunits. Nitrilase exploits the Lys-Cys-Glu catalytic triad which is essential for its active site function and enhancing its performance. The structure of a thermoactive nitrilase