A high throughput screening strategy for the assessment of nitrile-hydrolyzing activity towards the production of enantiopure β-hydroxy acids

• We report a strategy to facilitate rapid identification of bacterial isolates demonstrating nitrile hydrolysing activity.
• This incorporates toxicity, starvation and induction studies along with subsequent colorimetric screening for activity.
• This has enabled the rapid biocatalytic screening of 256 novel bacterial isolates towards β-hydroxynitriles.
• A whole cell catalyst SET-1 was identified and catalysed the hydrolysis of 3-hydroxybutyronitrile with remarkably high ee.
• (S)-3-hydroxybutyric acid was produced under mild conditions in 42% yield and >99.9% ee.

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.

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