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-methylglutaronit rile as the sole nitrogen source. Enzyme expression is constitutive an d 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 1 0.3 mu mole h(-1) mg wet cells(-1). The enzyme in cell-free preparatio ns is inhibited by a number of heavy metals, phenylhydrazine, and cyan ide. Substrate specificity is broad with highest rates shown on C-4 an d C-5 aliphatic mononitriles. The strain appears somewhat unusual in i ts dependence on cobalt supplementation for maximum enzyme activity an d 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.