Synthesis of ( R )-mandelic acid and ( R )-mandelic acid amide by recombinant E. coli strains expressing a ( R )-specifi
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ORIGINAL RESEARCH PAPER
Synthesis of (R)-mandelic acid and (R)-mandelic acid amide by recombinant E. coli strains expressing a (R)-specific oxynitrilase and an arylacetonitrilase Erik Mu¨ller . Olga Sosedov . Janosch Alexander David Gro¨ning . Andreas Stolz
Received: 9 April 2020 / Accepted: 3 September 2020 Ó The Author(s) 2020
Abstract Objectives Chiral 2-hydroxycarboxylic acids and 2-hydroxycarboxamides are valuable synthons for the chemical industry. Results The biocatalytic syntheses of (R)-mandelic acid and (R)-mandelic acid amide by recombinant Escherichia coli clones were studied. Strains were constructed which simultaneously expressed a (R)specific oxynitrilase (hydroxynitrile lyase) from the plant Arabidopsis thaliana together with the arylacetonitrilase from the bacterium Pseudomonas fluorescens EBC191. In addition, recombinant strains were constructed which expressed a previously described acid tolerant variant of the oxynitrilase and an amide forming variant of the nitrilase. The whole cell catalysts which simultaneously expressed E. Mu¨ller O. Sosedov J. A. D. Gro¨ning A. Stolz (&) Institut fu¨r Mikrobiologie, Universita¨t Stuttgart, Allmandring 31, 70569 Stuttgart, Germany e-mail: [email protected] E. Mu¨ller e-mail: [email protected] O. Sosedov e-mail: [email protected] J. A. D. Gro¨ning e-mail: [email protected] Present Address: O. Sosedov Biochem Labor fu¨r chemische Analytik GmbH, Daimlerstr. 5B, 76185 Karlsruhe, Germany
the (R)-specific oxynitrilase and the wild-type nitrilase transformed in slightly acidic buffer systems benzaldehyde plus cyanide preferentially to (R)-mandelic acid with ee-values [ 95%. The combination of the (R)-specific oxynitrilase with the amide forming nitrilase variant gave whole cell catalysts which converted at pH-values B pH 5 benzaldehyde plus cyanide with a high degree of enantioselectivity (ee [ 90%) to (R)-mandelic acid amide. The acid and the amide forming catalysts also converted chlorinated benzaldehydes with cyanide to chlorinated mandelic acid or chlorinated mandelic acid amides. Conclusions Efficient systems for the biocatalytic production of (R)-2-hydroxycarboxylic acids and (R)2-hydroxycarboxamides were generated. Keywords Biotransformations Chiral synthesis Nitrilase Oxynitrilase Hydroxynitrile lyase Enzyme cascades
Introduction Organic nitriles are natural products and are also synthesized in huge amounts by the chemical industry. There are several enzymes known which either form or convert organic nitriles and there is considerable interest in applying these enzymes in biotechnology. Currently, the major interest in nitrile forming enzyme
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lies in oxynitrilases (hydroxynitrile lyases) which can form chiral 2-hydroxynitriles from aldehydes (or ketones) and cyanide (Bracco et al. 2016). In addition, there are two groups of nitriles converting enzymes intensively studied which possess a considerable potential for bio
