Nitrile hydrolysing enzymes have found wide use in the pharmaceutical industry for the production of fine chemicals. This work presents a strategy that facilitates the rapid identification of bacterial isolates demonstrating nitrile hydrolysing activity. The strategy incorporates toxicity, starvation and induction studies along with subsequent colorimetric screening for activity, further focusing the assessment towards the substrates of interest. This high-throughput strategy uses a 96 well plate system, and has enabled the rapid biocatalytic screening of 256 novel bacterial isolates towards β- hydroxynitriles. Results demonstrate the strategy’s potential to rapidly assess a variety of β-hydroxynitriles including aliphatic, aromatic and dinitriles. A whole cell catalyst Rhodococcus erythropolis SET1 was identified and found to catalyse the hydrolysis of 3-hydroxybutyronitrile with remarkably high enantioselectivity under mild conditions, to afford (S)-3-hydroxybutyric acid in 42% yield and >99.9% ee. The biocatalytic capability of this strain including the variation of parameters such as temperature and time were further investigated and all results indicate the presence of a highly enantioselective if not enantiospecific nitrilase enzyme within the microbial whole cell. We present substrate evaluation with 34 chiral nitriles of Rhodococcus erythropolis SET1. These substrates consist primarily of β-hydroxy nitriles with varying alkyl and aryl groups at the β-position containing in some cases, various substituents at the α- position. In the case of β-hydroxy nitriles unsubstituted at the α-position, acids were the major products as a result of suspected nitrilase activity of the isolate. Unexpectedly, amides were found to be the major products when β-hydroxynitriles were substituted at the α-position with a vinyl group. Therefore this novel isolate has demonstrated additional NHase behaviour which is dependent on the functionality at the α-position. In order to probe this mechanism further related substrates were evaluated and amide was observed where other electron withdrawing groups were present at the α-position. Additionally various parameters which may influence the biocatalytic hydrolysis by SET1 were studied and are presented herein.
|Publication status||Unpublished - 2014|
- Hydrolysing enzymes, Organic synthesis