Application of a Recombinant Escherichia coli Whole‐Cell Catalyst Expressing Hydroxynitrile Lyase and Nitrilase Activities in Ionic Liquids for the Production of (S)‐Mandelic Acid and (S)‐Mandeloamide

Abstract

AbstractThe conversion of benzaldehyde and cyanide into mandelic acid and mandeloamide by a recombinant Escherichia coli strain which simultaneously expressed an (S)‐hydroxynitrile lyase (oxynitrilase) from cassava (Manihot esculenta) and an arylacetonitrilase from Pseudomonas fluorescens EBC191 was studied. Benzaldehyde exhibited a pronounced inhibitory effect on the nitrilase activity in concentrations ≥25 mM. Therefore, it was tested if two‐phase systems consisting of a buffered aqueous phase and the ionic liquid 1‐butyl‐1‐pyrrolidinium bis(trifluoromethanesulfonyl)imide (BMpl NTf2) or 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMim PF6) could be used for the intended biotransformation. The distribution coefficients of the substrates, intermediates and products of the reaction were determined and it was found that BMpl NTf2 and BMim PF6 were highly efficient as substrate reservoirs for benzaldehyde. The recombinant E. coli strain was active in the presence of BMpl NTf2 or BMim PF6 phases and converted benzaldehyde and cyanide into mandelic acid and mandeloamide. The two‐phase systems allowed the conversion of benzaldehyde dissolved in the ionic liquids to a concentration of 700 mM with product yields (=sum of mandelic acid and mandeloamide) of 87–100%. The cells were slightly more effective in the presence of BMpl NTf2 than in the presence of BMim PF6. In both two‐phase systems benzaldehyde and cyanide were converted into (S)‐mandeloamide and (S)‐mandelic acid with enantiomeric excesses ≥94%. The recombinant E. coli cells formed, in the two‐phase systems with ionic liquids and increased substrate concentrations, higher relative amounts of mandeloamide than in a purely aqueous system with lower substrate concentrations.