Conversion of sterically demanding alpha,alpha-disubstituted phenylacetonitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191
| dc.contributor.author | Baum, Stefanie | |
| dc.contributor.author | Williamson, Dael S | |
| dc.contributor.author | Sewell, Trevor | |
| dc.contributor.author | Stolz, Andreas | |
| dc.date.accessioned | 2016-09-01T10:16:11Z | |
| dc.date.available | 2016-09-01T10:16:11Z | |
| dc.date.issued | 2012 | |
| dc.date.updated | 2016-09-01T10:14:36Z | |
| dc.description.abstract | 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. | en_ZA |
| dc.identifier | http://dx.doi.org/10.1128/AEM.05570-11 | |
| dc.identifier.apacitation | Baum, S., Williamson, D. S., Sewell, T., & Stolz, A. (2012). Conversion of sterically demanding alpha,alpha-disubstituted phenylacetonitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191. <i>Applied and Environmental Microbiology</i>, http://hdl.handle.net/11427/21641 | en_ZA |
| dc.identifier.chicagocitation | Baum, Stefanie, Dael S Williamson, Trevor Sewell, and Andreas Stolz "Conversion of sterically demanding alpha,alpha-disubstituted phenylacetonitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191." <i>Applied and Environmental Microbiology</i> (2012) http://hdl.handle.net/11427/21641 | en_ZA |
| dc.identifier.citation | Baum, S., Williamson, D. S., Sewell, T., & Stolz, A. (2012). Conversion of Sterically Demanding α, α-Disubstituted Phenylacetonitriles by the Arylacetonitrilase from Pseudomonas fluorescens EBC191. Applied and environmental microbiology, 78(1), 48-57. | en_ZA |
| dc.identifier.issn | 0099-2240 | en_ZA |
| dc.identifier.ris | TY - Journal Article AU - Baum, Stefanie AU - Williamson, Dael S AU - Sewell, Trevor AU - Stolz, Andreas AB - 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. DA - 2012 DB - OpenUCT DP - University of Cape Town J1 - Applied and Environmental Microbiology LK - https://open.uct.ac.za PB - University of Cape Town PY - 2012 SM - 0099-2240 T1 - Conversion of sterically demanding alpha,alpha-disubstituted phenylacetonitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191 TI - Conversion of sterically demanding alpha,alpha-disubstituted phenylacetonitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191 UR - http://hdl.handle.net/11427/21641 ER - | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11427/21641 | |
| dc.identifier.uri | http://aem.asm.org/content/78/1/48.short | |
| dc.identifier.vancouvercitation | Baum S, Williamson DS, Sewell T, Stolz A. Conversion of sterically demanding alpha,alpha-disubstituted phenylacetonitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191. Applied and Environmental Microbiology. 2012; http://hdl.handle.net/11427/21641. | en_ZA |
| dc.language | eng | en_ZA |
| dc.publisher | American Society for Microbiology | en_ZA |
| dc.publisher.institution | University of Cape Town | |
| dc.source | Applied and Environmental Microbiology | en_ZA |
| dc.source.uri | http://aem.asm.org/ | |
| dc.subject.other | alpha | |
| dc.subject.other | alpha-disubstituted phenylacetonitriles | |
| dc.subject.other | arylacetonitrilase | |
| dc.subject.other | Pseudomonas fluorescens EBC191 | |
| dc.title | Conversion of sterically demanding alpha,alpha-disubstituted phenylacetonitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191 | en_ZA |
| dc.type | Journal Article | en_ZA |
| uct.type.filetype | Text | |
| uct.type.filetype | Image | |
| uct.type.publication | Research | en_ZA |
| uct.type.resource | Article | en_ZA |