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

Abstract

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 rhamnose-inducible promoter. The whole cell catalysts obtained converted benzaldehyde 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 in 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 the (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%.

Conversion of Sterically Demanding α,α-Disubstituted Phenylacetonitriles by the Arylacetonitrilase from Pseudomonas fluorescens EBC191

“…The analysis of the chemically synthesized atrolactamide by chiral HPLC (conditions described above) demonstrated the presence of two signals (retention time [R t ] of 2.3 min and R t of 3.8 min). In order to correlate these signals with the respective enantiomers, acetophenone plus cyanide were converted by E. coli JM109(pIK9)(pJOE5361.1), which simultaneously expressed the (S)-oxynitrilase from cassava (Manihot esculenta) and the nitrilase from P. fluorescens EBC191 and which had been successfully used for the production of (S)-mandelic acid and (S)-mandeloamide from benzaldehyde plus cyanide (40). Thus, it was found that during the reaction, the signal with an R t of 3.8 min was formed preferentially, which was thus identified as (S)-atrolactamide.…”

“…Escherichia coli JM109 was used as the host strain for all plasmids. The cultivation and induction conditions for the recombinant strains used were described previously (16,20,40). Enzyme assays.…”

“…A possible solution for this problem has recently been achieved by constructing "bienzymatic cascade reactions," which couple a highly (S)-specific plant-derived oxynitrilase with a bacterial nitrilase either in vitro (as immobilized enzyme catalysts) or in vivo (in recombinant whole-cell catalysts). These systems convert benzaldehyde plus cyanide efficiently to (S)-mandelic acid and/or (S)-mandeloamide (29,40,41). These biotransformation routes are interesting, as (R)-and (S)-mandelic acids are utilized in various chemical syntheses as convenient precursors for the introduction of a chiral center, which possess the extra advantage of bearing a useful functional group.…”

“…Therefore, it might be expected that a combination of enantioselective oxynitrilases with bacterial (or fungal) nitrilases (or nitrile hydratases) should result in the generation of broadly applicable biocatalysts with the ability to convert a wide range of aldehydes (and ketones) plus cyanide to chiral 2-hydroxycarboxylic acids or 2-hydroxycarboxamides, which are important intermediates in chemical synthesis (8). Previously performed experiments aimed exclusively for the formation of (S)-mandelic acid and/or (S)-mandeloamide from benzaldehyde and cyanide (29,40,41), but there exist several other relevant targets for this kind of biotransformation. A very interesting group of products is ␣-alkyl-␣-hydroxycarboxylic acids (and amides), which can be synthesized by a combination of oxynitrilases and nitrilases from different ketones and cyanide (Fig.…”

The influence of hydroxypropyl-β-cyclodextrin on the enantioselective hydrolysis of 2-amino phenylpropionitrile catalyzed by recombinant nitrilase

“…For example, (R)-(−)-mandelic acid, widely used for the production of semisynthetic cephalosporins and anti-obesity agents, is produced by hydrolyzing mandelonitrile [5,8]. In addition, other studies focused on strategic optimizations for increasing the reaction rate, reducing substrate inhibition, and improving enzyme enantioselectivity [9][10][11].…”