Construction of Recombinant Escherichia coli Catalysts which Simultaneously Express an (S)-Oxynitrilase and Different Nitrilase Variants for the Synthesis of (S)-Mandelic Acid and (S)-Mandelic Amide from Benzaldehyde and Cyanide

Recombinant Escherichia coli strains were constructed which simultaneously expressed the genes encoding the (S)-oxynitrilase from cassava (Manihot esculenta) together with the wild-type or a mutant variant of the arylacetonitrilase from Pseudomonas fluorescens EBC191 in a single organism under the control of a rharnnose-inducible promoter. The whole cell catalysts obtained converted benzal-dehyde and potassium cyanide in aqueous media at pH 5.2 mainly to (S)-mandelic acid and/or (S)-mandelic amide and synthesized only low amounts of the corresponding (R)-enantiomers. The conversion of benzaldehyde and potassium cyanide (KCN) by a whole-cell catalyst simultaneously expressing, the (S)oxynitrilase and the wild-type nitrilase resulted ill a ratio of (S)-mandelic acid to (S)-mandelic amide of about 4:3. This could be explained by the strong nitrile hydratase activity of the wild-type nitrilase with (S)-mandelonitrile as substrate. The relative proportion of (S)-mandelic amide formed in this system was significantly increased by coexpressing file (S)-oxynitrilase with a carboxy-terminally truncated variant of the nitrilase. This whole-cell catalyst Converted benzaldehyde and KCN to mandelic amide and mandelic acid in a ratio of about 9:1. The ee of the (S)-mandelic amide formed Was calculated to be >95%.