Screening, characterization and application of cyanide-resistant nitrile hydratases

Two new bacterial strains, Pseudomonas marginales MA32 and Pseudomonas putida MA113, containing nitrile hydratases resistant to cyanide were isolated from soil samples by an enrichment procedure. In contrast to known nitrile hydratases, which rapidly lose activity at low to moderate cyanide concentrations, the enzymes described in this paper tolerate up to 50 mM cyanide. They show a broad substrate spectrum including not only small substrates like acrylonitrile but also nitriles with longer side chains and even nitriles with quarternary alpha-carbon atoms. Both characteristics are essential for the transformation of ketone cyanohydrins, which are much more instable and therefore releasing much higher amounts of prussic acid than cyanohydrins formed from aldehydes. P. marginales MA32 was used as a whole cell biocatalyst for the hydration of acetone cyanohydrin to alpha-Hydroxyisobutyramide, which is a precursor of methacrylamide, an important pre-polymer. After optimization of the process conditions a maximum amide concentration of more than 1.6 M could be reached within 5 hours with 5 g/L biocatalyst referred to cell dry weight.