Simultaneous expression of an arylacetonitrilase from Pseudomonas fluorescens and a (S)-oxynitrilase from Manihot esculenta in Pichia pastoris for the synthesis of (S)-mandelic acid

The arylacetonitrilase of Pseudomonas fluorescens EBC191 catalyzes the conversion of (S)-mandelonitrile to (S)-mandelic acid and (S)-mandeloamide. This biotransformation is optimally performed under acidic pH values because (S)-mandelonitrile rapidly decomposes under neutral conditions. Therefore, the gene encoding the arylacetonitrilase of P. fluorescens EBC191 was integrated and expressed under the control of the AOX1 promoter in the methylotrophic yeast Pichia pastoris which was supposed to act as an acidotolerant expression system. These recombinant strains hydrolyzed (R,S)-mandelonitrile at pH values >or=3 to mandelic acid and mandeloamide and were more acidotolerant than previously constructed Escherichia coli whole cell catalysts synthesizing the same nitrilase activity. Subsequently, recombinant P. pastoris strains were constructed which simultaneously expressed the (S)-oxynitrilase of Manihot esculenta and the arylacetonitrilase of P. fluorescens EBC191 each under the control of individual AOX1 promoters in order to obtain a whole cell catalyst for the synthesis of (S)-mandelic acid from benzaldehyde and cyanide. Resting cells of the recombinant strains converted under acidic conditions benzaldehyde and cyanide initially to mandelonitrile which was immediately converted to mandelic acid and mandeloamide. The chiral analysis of the products formed revealed a high enantiomeric excess for the (S)-enantiomers.     

Enzymatic synthesis of (R)-cyanohydrins by a novel (R)-oxynitrilase from Vicia sativa L.

The defatted seed meal of common vetch (Vicia sativa L.) served as a source of (R)-oxynitrilase which catalyzed the enantioselective addition of HCN to aromatic, heteroaromatic, fluoro-substituted aromatic aldehydes to produce the corresponding enantiomeric pure cyanohydrins in yields of 52–100% and 88–99% ee at 12°C under micro-aqueous medium (diisopropyl ether) without addition of buffer solution.     

Enzymatic formation and esterification of (S)-mandelonitrile

The (S)-selective hydroxynitrile lyase from Hevea brasiliensis (HbHNL) catalyzes the trans-cyanohydrin reaction (transcyanation). The equilibrium of this two-step reaction sequence is not favorable unless a large excess of acetone cyanohydrin (1) is used. Therefore, the coupling of this reaction with a follow-up reaction was investigated. It was established that the trans-cyanohydrin reaction could be performed in organic media, making it possible to couple it with a lipase-catalyzed acylation. Candida antarctica lipase B (CAL-B) shows a high selectivity (E=100) for (S)-mandelonitrile (4) and is, therefore, the ideal candidate for this type of multi-step one-pot reaction.