Isolation of glycolonitrile-hydrolyzing microorganism based on colorimetric reaction

To isolate glycolonitrile-hydrolyzing microorganisms, a simple and rapid high-throughput screening method based on a colorimetric reaction of glycolic acid with β-naphthol in sulfuric acid solution was developed. Four strains able to convert glycolonitrile to glycolic acid were isolated from soil samples using this screening method, among which Rhodococcus sp. ZJUT-N595 displayed the highest hydrolytic activity. Resting cells of Rhodococcus sp. ZJUT-N595 could also biotransform acrylonitrile, racemic 2,2-dimethylcyclopropanecarbonitrile to corresponding amides as well as carboxylic acids, and racemic 2,2-dimethylcyclopropanecarboxamide to (S)-2,2-dimethylcyclopropanecarboxlic acid (e.e. > 90%), which demonstrated that this strain contained both intracellular nitrile hydratase and amidase activities. Batch cultivation showed that the optimal growth temperature and initial medium pH for Rhodococcus sp. ZJUT-N595 glycolonitrile-hydrolyzing activity were 30 °C and pH 8, respectively. The preferred carbon and nitrogen source and ideal inducer were mannitol, a composite of peptone plus yeast extract and caprolactam.