Michihiko Kobayashi

The catalytic mechanism of amidase also involves nitrile hydrolysis

The amidase from Rhodococcus rhodochrous J1, which hydrolyzes an amide to an acid and ammonium, was surprisingly found to catalyze the hydrolytic cleavage of the C‐N triple bond in a nitrile to form an acid and ammonium stoichiometrically. The amidase exhibited a K m of 3.26 mM for benzonitrile in contrast to that of 0.15 mM for benzamide as the original substrate, but the V max for benzonitrile was about 1/6000 of that for benzamide. A mutant amidase containing alanine instead of Ser195, which is essential for amidase catalytic activity, showed no nitrilase activity, demonstrating that this residue plays a crucial role in the hydrolysis of nitriles as well as amides.

Hyperinduction of nitrile hydratase acting on indole-3-acetonitrile in Agrobacterium tumefaciens

We investigated the optimum conditions for the formation of nitrile hydratase (NHase), which acts on indole-3-acetonitrile, in Agrobacterium tumefaciens. Good inducers for enzyme formation have been found to be roughly classified into three representative types of amides such as pivalamide, crotonamide and e-caprolactam. When the strain was cultivated in the optimum culture medium containing ε-caprolactam as an inducer, in particular, the specific activity of NHase in the culture was 13 000 times higher than that without addition of amides, nitriles or acids. In this case, NHase formed accounted for 12% of the total cellular soluble protein. The purified NHase did not act on ε-caprolactam, and ε-caprolactam was not degraded during the cultivation by the strain, suggesting that ε-caprolactam seems to keep driving the NHase induction mechanism.