Nitrilase activity analysis

All nitrilases belonging to aromatic nitrilase and arylacetonitrilase are susceptible to SH-reagents, except the R. rhodochrous K22 aliphatic nitrilase [81]. Analysis by site-directed mutagenesis for our three nitrilases from R. rhodochrous J1 [83], R. rhodochrous K22 [84] and Alcaligenes faecalis JM3 [85] revealed that a unique cysteine that is conserved at the corresponding position in each nitrilase is essential for the catalytic activity (Fig. 7). All nitrilases whose structural genes are cloned have a similar amino acid sequence nitrilase forms a superfamily. 

An enantioselective nitrilase from Pseudomonas putida isolated from soil cultured with 2 mM phenylacetonitrile was purified and characterized. This enzyme is comprised of 9-10 identical subunits each of 43 kDa. It exhibits a pH optimum at 7.0 and a temperature optimum at 40 °C (Ty2 = 160 min) and requires a reducing environment for activity. This nitrilase was shown to have an unusually high tolerance for acetone as co-solvent, with >50% activity retained in the presence of 30% acetone. The kinetic profile of this nitrilase reveals KM= 13.4mM, cat/ M = 0-9s 1mM 1 for mandelonitrile, ZfM = 3.6mM, kclJKM 5.2 s him-1 for phenylacetonitrile, and KM = 5.3 mM, kC lt/KM = 2.5 s 1 him 1 for indole 3-acetonitrile. Preliminary analysis of this enzyme with 5 mM mandelonitrile revealed formation of (/t)-mandelic acid with 99.9% ee [59]. 

Analysis of the reaction products showed that during conversion of succinonitrile into 3-cyanopropionic acid, succinamic acid (H2NOC-CH2-CH2-COOH) was detected as a free intermediate in the reaction mixture, suggesting that enzymatic activities other than nitrilases (i.e. nitrile hydratase) were present in the cell... 

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