Comparative Analysis of the Conversion of Mandelonitrile and 2-Phenylpropionitrile by a Large Set of Variants Generated from a Nitrilase Originating from Pseudomonas fluorescens EBC191

The arylacetonitrilase from the bacterium EBC191 has been intensively studied as a model to understand the molecular basis for the substrate-, reaction-, and enantioselectivity of nitrilases. The nitrilase converts various aromatic and aliphatic nitriles to the corresponding acids and varying amount...

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Bibliographic Details
Published in:Molecules (Basel, Switzerland) Switzerland), 2019-11, Vol.24 (23), p.4232
Main Authors: Stolz, Andreas, Eppinger, Erik, Sosedov, Olga, Kiziak, Christoph
Format: Article
Language:English
Subjects:
Acetonitriles - metabolism
Amides
Amino acids
Aminohydrolases - genetics
Aminohydrolases - metabolism
Aqueous solutions
Arylacetonitrilase
Binding sites
Comparative analysis
Conversion
Cyanides
Enantiomers
enantioselectivity
Enzymes
Mandelonitrile
Microscopy
Mutagenesis
Mutation
Nitrilase
Nitriles
Nitrogen
Pseudomonas fluorescens
Pseudomonas fluorescens - enzymology
reaction specificity
Review
structure-function relationship
Substrate Specificity
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Summary:The arylacetonitrilase from the bacterium EBC191 has been intensively studied as a model to understand the molecular basis for the substrate-, reaction-, and enantioselectivity of nitrilases. The nitrilase converts various aromatic and aliphatic nitriles to the corresponding acids and varying amounts of the corresponding amides. The enzyme has been analysed by site-specific mutagenesis and more than 50 different variants have been generated and analysed for the conversion of ( , )-mandelonitrile and ( , )-2-phenylpropionitrile. These comparative analyses demonstrated that single point mutations are sufficient to generate enzyme variants which hydrolyse ( , )-mandelonitrile to ( )-mandelic acid with an enantiomeric excess (ee) of 91% or to ( )-mandelic acid with an ee-value of 47%. The conversion of ( , )-2-phenylpropionitrile by different nitrilase variants resulted in the formation of either ( )- or ( )-2-phenylpropionic acid with ee-values up to about 80%. Furthermore, the amounts of amides that are produced from ( , )-mandelonitrile and ( , )-2-phenylpropionitrile could be changed by single point mutations between 2%-94% and
ISSN:1420-3049
1420-3049
DOI:10.3390/molecules24234232