A Pseudomonas putida capable of stereoselective hydrolysis of nitriles

Pseudomonas putida NRRL-18668 contains a nitrile hydratase capable of stereoselective hydrolysis of 2-(4-chlorophenyl)-3-methylbutyronitrile at more than 90% enantiomeric excess (ee) to the (S)-amide. This soil isolate was recovered from enrichments using (R,S)-2-methylglutaronitrile as the sole nitrogen source. Enzyme expression is constitutive and does not show a high level of catabolite repression. The organism is capable of growth on a wide variety of aliphatic mono- and dinitrile compounds. The hydrolysis activity on propionitrile is approximately 10.3 mu mole h(-1) mg wet cells(-1). The enzyme in cell-free preparations is inhibited by a number of heavy metals, phenylhydrazine, and cyanide. Substrate specificity is broad with highest rates shown on C-4 and C-5 aliphatic mononitriles. The strain appears somewhat unusual in its dependence on cobalt supplementation for maximum enzyme activity and the ability to hydrolyze some aromatic nitriles. This strain is also capable of a two-step hydrolysis of 2-(4-isobutylphenyl)propionitrile and 2-(6-methoxy-2-napthyl)-propionitrile to the (S)-acids (ibuprofen and naproxen respectively) with stereoselectivity residing primarily in the aliphatic amidase. This appears to be the first description of a steroselective nitrile hydratase from a gram-negative organism.