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

• Main Authors: Andreas Stolz, Erik Eppinger, Olga Sosedov, Christoph Kiziak
• Format: Article
• Language: English
• Published: MDPI AG 2019-11-01
• Series: Molecules
• Subjects: nitrilase; reaction specificity; enantioselectivity; structure-function relationship
• Online Access: https://www.mdpi.com/1420-3049/24/23/4232

The arylacetonitrilase from the bacterium <i>Pseudomonas fluorescens</i> 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 (<i>R</i>,<i>S</i>)-mandelonitrile and (<i>R</i>,<i>S</i>)-2-phenylpropionitrile. These comparative analyses demonstrated that single point mutations are sufficient to generate enzyme variants which hydrolyse (<i>R</i>,<i>S</i>)-mandelonitrile to (<i>R</i>)-mandelic acid with an enantiomeric excess (ee) of 91% or to (<i>S</i>)-mandelic acid with an ee-value of 47%. The conversion of (<i>R</i>,<i>S</i>)-2-phenylpropionitrile by different nitrilase variants resulted in the formation of either (<i>S</i>)- or (<i>R</i>)-2-phenylpropionic acid with ee-values up to about 80%. Furthermore, the amounts of amides that are produced from (<i>R</i>,<i>S</i>)-mandelonitrile and (<i>R</i>,<i>S</i>)-2-phenylpropionitrile could be changed by single point mutations between 2%&#8722;94% and &lt;0.2%&#8722;73%, respectively. The present study attempted to collect and compare the results obtained during our previous work, and to obtain additional general information about the relationship of the amide forming capacity of nitrilases and the enantiomeric composition of the products.