Geobacillus pallidus RAPc8 (NRRL: B-59396) is a moderately thermophilic gram-positive bacterium, originally isolated from Australian lake sediment. The G. pallidus RAPc8 gene encoding an inducible nitrilase was located and cloned using degenerate primers coding for well-conserved nitrilase sequences, coupled with inverse PCR. The nitrilase open reading frame was cloned into an expression plasmid and the expressed recombinant enzyme purified and characterized. The protein had a monomer molecular weight of 35,790 Da, and the purified functional enzyme had an apparent molecular weight of approximately 600 kDa by size exclusion chromatography. Similar to several plant nitrilases and some bacterial nitrilases, the recombinant G. pallidus RAPc8 enzyme produced both acid and amide products from nitrile substrates. The ratios of acid to amide produced from the substrates we tested are significantly different to those reported for other enzymes, and this has implications for our understanding of the mechanism of the nitrilases which may assist with rational design of these enzymes. Electron microscopy and image classification showed complexes having crescent-like, "c-shaped", circular and "figure-8" shapes. Protein models suggested that the various complexes were composed of 6, 8, 10 and 20 subunits, respectively.

The nitrilase from Pseudomonas fluorescens EBC191 converted 2-methyl-2-phenylpropionitrile, which contains a quaternary carbon atom in the ␣-position toward the nitrile group, and also similar sterically demanding substrates, such as 2-hydroxy-2-phenylpropionitrile (acetophenone cyanohydrin) or 2-acetyloxy-2-methylphenylacetonitrile. 2-Methyl-2-phenylpropionitrile was hydrolyzed to almost stoichiometric amounts of the corresponding acid. Acetophenone cyanohydrin was transformed to the corresponding acid (atrolactate) and amide (atrolactamide) at a ratio of about 3.4:1. The (R)-acid and the (S)-amide were formed preferentially from acetophenone cyanohydrin. A homology model of the nitrilase suggested that steric hindrance with amino acid residue Tyr54 could impair the binding or conversion of sterically demanding substrates. Therefore, several enzyme variants that carried mutations in the respective residues were generated and subsequently analyzed for the substrate specificity and enantioselectivity of the reactions. Enzyme variants that demonstrated increased relative activities for the conversion of acetophenone cyanohydrin were identified. The chiral analysis of these reactions demonstrated peculiar reaction kinetics, which suggested that the enzyme variants converted the nonpreferred (S)-enantiomer of acetophenone cyanohydrin with a higher reaction rate than that of the (preferred) (R)-enantiomer. Recombinant whole-cell catalysts that simultaneously produced the nitrilase from P. fluorescens EBC191 and a plant-derived (S)-oxynitrilase from cassava (Manihot esculenta) converted acetophenone plus cyanide at pH 4.5 to (S)-atrolactate and (S)-atrolactamide. These recombinant cells are promising catalysts for the synthesis of stable chiral quaternary carbon centers from ketones.