Synthesis of enantiomerically pure (S)-mandelic acid using an oxynitrilase–nitrilase bienzymatic cascade: a nitrilase surprisingly shows nitrile hydratase activity

Mateo, César; Chmura, A.; Rustler, Sven; Rantwijk, Fred van; Stolz, A.; Sheldon, Roger A.

doi:10.1016/j.tetasy.2006.01.020

Cited by 143 publications

(87 citation statements)

References 12 publications

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“…22 Only nitrilases are commercially available, but occasional formation of amide in nitrilase-catalyzed hydolysis of nitriles has been observed. 23 This partial hydrolysis may depend on the structure of the nitrile, as in the case reported for a nitrilasecatalyzed hydrolysis of a β-hydroxynitrile. 24 We have used the commercially available nitrilase from Arabidopsis thaliana and carried out the reaction in a 10% DMSO aqueous solution of the nitrile 3a.…”

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confidence: 92%

2,6-Disubstituted benzothiazoles, analogues of the aromatic core of D-luciferin: synthesis and evaluation of the affinity for Photinus pyralis luciferase

Meroni¹,

Ciana²,

Meda³

et al. 2009

Arkivoc

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confidence: 92%

2,6-Disubstituted benzothiazoles, analogues of the aromatic core of D-luciferin: synthesis and evaluation of the affinity for Photinus pyralis luciferase

Meroni¹,

Ciana²,

Meda³

et al. 2009

Arkivoc

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“…This enzyme converted various phenylacetonitriles (e.g., 2-PPN, Oacetoxymandelonitrile, or mandelonitrile), and also aliphatic 2-acetoxynitriles, with moderate enantioselectivities into the corresponding ␣-substituted carboxylic acids. Furthermore, with some substrates, significant amounts of the corresponding amides were also formed (5,8,12,21,27).…”

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confidence: 99%

Identification of Amino Acid Residues Responsible for the Enantioselectivity and Amide Formation Capacity of the Arylacetonitrilase fromPseudomonas fluorescensEBC191

Kiziak

2009

Appl Environ Microbiol

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The nitrilase from Pseudomonas fluorescens EBC191 converted (R,S)-mandelonitrile with a low enantioselectivity to (R)-mandelic acid and (S)-mandeloamide in a ratio of about 4:1. In contrast, the same substrate was hydrolyzed by the homologous nitrilase from Alcaligenes faecalis ATCC 8750 almost exclusively to (R)-mandelic acid. A chimeric enzyme between both nitrilases was constructed, which represented in total 16 amino acid exchanges in the central part of the nitrilase from P. fluorescens EBC191. The chimeric enzyme clearly resembled the nitrilase from A. faecalis ATCC 8750 in its turnover characteristics for (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile (2-PPN) and demonstrated an even higher enantioselectivity for the formation of (R)-mandelic acid than the nitrilase from A. faecalis. An alanine residue (Ala165) in direct proximity to the catalytically active cysteine residue was replaced in the nitrilase from P. fluorescens by a tryptophan residue (as found in the nitrilase from A. faecalis ATCC 8750 and most other bacterial nitrilases) and several other amino acid residues. Those enzyme variants that possessed a larger substituent in position 165 (tryptophan, phenylalanine, tyrosine, or histidine) converted racemic mandelonitrile and 2-PPN to increased amounts of the R enantiomers of the corresponding acids. The enzyme variant Ala165His showed a significantly increased relative activity for mandelonitrile (compared to 2-PPN), and the opposite was found for the enzyme variants carrying aromatic residues in the relevant position. The mutant forms carrying an aromatic substituent in position 165 generally formed significantly reduced amounts of mandeloamide from mandelonitrile. The important effect of the corresponding amino acid residue on the reaction specificity and enantiospecificity of arylacetonitrilases was confirmed by the construction of a Trp164Ala variant of the nitrilase from A. faecalis ATCC 8750. This point mutation converted the highly R-specific nitrilase into an enzyme that converted (R,S)-mandelonitrile preferentially to (S)-mandeloamide.

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“…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.…”

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confidence: 99%

“…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.…”

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

Baum

Williamson

Sewell

2012

Appl Environ Microbiol

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The nitrilase from Pseudomonas fluorescens EBC191 converted 2-methyl-2-phenylpropionitrile, which contains a quaternary carbon atom in the ␣-position toward the nitrile group, and also similar sterically demanding substrates, such as 2-hydroxy-2-phenylpropionitrile (acetophenone cyanohydrin) or 2-acetyloxy-2-methylphenylacetonitrile. 2-Methyl-2-phenylpropionitrile was hydrolyzed to almost stoichiometric amounts of the corresponding acid. Acetophenone cyanohydrin was transformed to the corresponding acid (atrolactate) and amide (atrolactamide) at a ratio of about 3.4:1. The (R)-acid and the (S)-amide were formed preferentially from acetophenone cyanohydrin. A homology model of the nitrilase suggested that steric hindrance with amino acid residue Tyr54 could impair the binding or conversion of sterically demanding substrates. Therefore, several enzyme variants that carried mutations in the respective residues were generated and subsequently analyzed for the substrate specificity and enantioselectivity of the reactions. Enzyme variants that demonstrated increased relative activities for the conversion of acetophenone cyanohydrin were identified. The chiral analysis of these reactions demonstrated peculiar reaction kinetics, which suggested that the enzyme variants converted the nonpreferred (S)-enantiomer of acetophenone cyanohydrin with a higher reaction rate than that of the (preferred) (R)-enantiomer. Recombinant whole-cell catalysts that simultaneously produced the nitrilase from P. fluorescens EBC191 and a plant-derived (S)-oxynitrilase from cassava (Manihot esculenta) converted acetophenone plus cyanide at pH 4.5 to (S)-atrolactate and (S)-atrolactamide. These recombinant cells are promising catalysts for the synthesis of stable chiral quaternary carbon centers from ketones.

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Synthesis of enantiomerically pure (S)-mandelic acid using an oxynitrilase–nitrilase bienzymatic cascade: a nitrilase surprisingly shows nitrile hydratase activity

Cited by 143 publications

References 12 publications

2,6-Disubstituted benzothiazoles, analogues of the aromatic core of D-luciferin: synthesis and evaluation of the affinity for Photinus pyralis luciferase

2,6-Disubstituted benzothiazoles, analogues of the aromatic core of D-luciferin: synthesis and evaluation of the affinity for Photinus pyralis luciferase

Identification of Amino Acid Residues Responsible for the Enantioselectivity and Amide Formation Capacity of the Arylacetonitrilase fromPseudomonas fluorescensEBC191

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

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