Construction and Application of Variants of the Pseudomonas fluorescens EBC191 Arylacetonitrilase for Increased Production of Acids or Amides

Abstract

The arylacetonitrilase from Pseudomonas fluorescens EBC191 differs from previously studied arylacetonitrilases by its low enantiospecificity during the turnover of mandelonitrile and by the large amounts of amides that are formed in the course of this reaction. In the sequence of the nitrilase from P. fluorescens, a cysteine residue (Cys163) is present in direct neighborhood (toward the amino terminus) to the catalytic active cysteine residue, which is rather unique among bacterial nitrilases. Therefore, this cysteine residue was exchanged in the nitrilase from P. fluorescens EBC191 for various amino acid residues which are present in other nitrilases at the homologous position. The influence of these mutations on the reaction specificity and enantiospecificity was analyzed with (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile as substrates. The mutants obtained demonstrated significant differences in their amide-forming capacities. The exchange of Cys163 for asparagine or glutamine residues resulted in significantly increased amounts of amides formed. In contrast, a substitution for alanine or serine residues decreased the amounts of amides formed. The newly discovered mutation was combined with previously identified mutations which also resulted in increased amide formation. Thus, variants which possessed in addition to the mutation Cys163Asn also a deletion at the C terminus of the enzyme and/or the modification Ala165Arg were constructed. These constructs demonstrated increased amide formation capacity in comparison to the mutants carrying only single mutations. The recombinant plasmids that encoded enzyme variants which formed large amounts of mandeloamide or that formed almost stoichiometric amounts of mandelic acid from mandelonitrile were used to transform Escherichia coli strains that expressed a plant-derived (S)-hydroxynitrile lyase. The whole-cell biocatalysts obtained in this way converted benzaldehyde plus cyanide either to (S)-mandeloamide or (S)-mandelic acid with high yields and enantiopurities.

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

“…Thus, Yeom and coworkers generated a model of the active site of the benzonitrilase from Rhodococcus rhodochrous ATCC 33278 and verified this model by the generation of mutants with modified activities for aliphatic nitriles or meta-substituted benzonitriles (54,55). Our group has recently demonstrated for the nitrilase from P. fluorescens EBC191 that large amino acid residues in the direct neighborhood of the catalytic active cysteine residue are a prerequisite for the preferred formation of (R)-mandelic acid from (R,S)-mandelonitrile (22,41).…”

“…A general reduction in the degree of amide formation was observed previously for other variants of the nitrilase from P. fluorescens EBC191. Thus, an exchange of the cysteine residue directly adjacent to the catalytically active cysteine residue (toward the amino terminus) by an alanine (Cys163Ala) or serine (Cys163Ser) residue or the introduction of a sterically demanding amino acid residue "on the other side" of the catalytically active cysteine residue (e.g., in Ala165Phe) resulted in a significant suppression of the nitrile hydratase activity of the nitrilase (22,41). The similar effects caused by mutations such as Cys163Ala, Ala165Phe, or Tyr54Ala on the reduction of amide formation in combination with the observed deleterious effect of other mutations at position 54 suggested that the amino acid residue Tyr54 is indeed located close to the catalytic center and thus indicate the correctness of the structural enzyme model.…”

“…1 2-Methyl-2-phenylpropionitrile and 2-methyl-2-phenylpropionic acid were purchased from Accela ChemBio Inc. (San Diego, CA), and 2-acetyloxy-2-methylphenylacetonitrile was kindly supplied by S. van Pelt, TU Delft (47). The sources of all other chemicals were described previously (41).…”

“…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.…”