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

The 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 aryl-A C H T U N G T R E N N U N G acetonitrilase 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 NTf 2 ) or 1-butyl-3-methylimidazolium hexafluorophosphate (BMim PF 6 ) 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 NTf 2 and BMim PF 6 were highly efficient as substrate reservoirs for benzaldehyde. The recombinant E. coli strain was active in the presence of BMpl NTf 2 or BMim PF 6 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 NTf 2 than in the presence of BMim PF 6 . 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 mandelo-A C H T U N G T R E N N U N G amide than in a purely aqueous system with lower substrate concentrations.

Efficient anti-Prelog enantioselective reduction of acetyltrimethylsilane to (R)-1-trimethylsilylethanol by immobilized Candida parapsilosis CCTCC M203011 cells in ionic liquid-based biphasic systems

“…Also, they may be explosive and are usually environmentally harmful. Ionic liquids (ILs), have recently emerged as novel green solvents for a great variety of biocatalytic transformations, and are becoming more and more attractive in such applications as a promising alternative to the conventional organic solvents [13-16]. Many kinds of ILs have proven to be biocompatible with diverse microbial cells, and present many advantages for the biotransformations such as high conversion rates, high enantioselectivity, excellent operational stability and recyclability [17].…”

Enzymatic cascade synthesis of (S)-2-hydroxycarboxylic amides and acids: Cascade reactions employing a hydroxynitrile lyase, nitrile-converting enzymes and an amidase

“…A further improvement in the enantiomeric purity of (S)-3 (from 94 to 98% at near-quantitative conversion of 1 and 2) was obtained by a using a combi-CLEA of MeHnL and PfNLase, prepared according to a suitably modified protocol [30,32]. A nearly complete conversion to ≥96% enantiomerically pure products was still obtained when the starting concentration of 1 was increased to 0.25 M. The yield of (S)-4 increased from 40% at 10 mM 1 to 58% at 0.25 M, which came as an unpleasant surprise but a similar trend was observed with a whole-cell biocatalyst [33]. The formation of major amounts of amide severely diminished the practical as well as the green value of our procedure and was addressed later.…”

“…present in asparagine, glutamine or arginine) close to the catalytic centre. Furthermore, also some point mutations in the positions 54, 110, 188 or 210 increased amide formation [33,45,46,[54][55][56]. In the course of our investigations, initially whole cell catalysts were constructed which simultaneously synthesised MeHnL together with a C-terminally truncated PfNLase variant.…”

“…Accordingly, the recombinant E. coli strain coexpressing the genes coding for MeHnL and PfNLase maintained its activity in biphasic aqueous BMPl NTf 2 or BMIm PF 6 and converted benzaldehyde plus HCN into mandelic acid and amide. These two-phase systems allowed the conversion of benzaldehyde dissolved in the ionic liquids to a concentration of 700 mM with combined product yields ((S)-3 and (S)-4) of 82-96% with enantiomeric excess ≥94% [33].…”

Whole-Cell Biocatalytic Processes with Ionic Liquids

“…3b). 95 6 ] were employed to form IL-containing biphasic systems, which systems allowed benzaldehyde to be dissolved in the IL phase to a concentration of 700 mM and thus to enable a high substrate conversion of 87-100%.…”