Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana . The nitrilase AtNIT1 from Arabidopsis thaliana was overexpressed in Escherichia coli with an N - terminal His6 tag and purified by zinc chelate affinity chromatography in a single step almost to homogeneity in a 68% yield with a specific activity of 34.1 U.mg-1 . The native enzyme ( approximately 450 kDa ) consists of 11-13 subunits ( 38 kDa ) . The temperature optimum was determined to be 35 degrees C and a pH optimum of 9 was found . Thus , recombinant AtNIT1 resembles in its properties the native enzyme and the nitrilase from Brassica napus . The stability of AtNIT1 could be significantly improved by the addition of dithiothreitol and EDTA . The substrate range of AtNIT1 differs considerably from those of bacterial nitrilases . Aliphatic nitriles are the most effective substrates , showing increasing rates of hydrolysis with increasing size of the residues , as demonstrated in the series butyronitrile , octanenitrile , phenylpropionitrile. In comparison with 3 - indolylacetonitrile , the rate of hydrolysis of 3 - phenylpropionitrile is increased by a factor of 330 , and the Km value is reduced by a factor of 23 . With the exception of fluoro , substituents in the alpha position to the nitrile function completely inhibit the hydrolysis.