Isolation Of α-Hydroxy Nitrile Hydrolyzing Microbes And Biotransformation Process For α-Hydroxy Acid Production

Using glycolonitrile to glycolic acid as a mode reaction, the presentpaper describes a high-throughput screening method forα-hydroxy nitrilehydrolyzing microbes able to be effectively used in the production ofα-hydroxy acid, with optimization of culture conditions andbiotransformation process.A simple and rapid HTS method based on a colorimetric reaction ofglycolic acid withβ-naphthol in sulfuric acid solution was developed. Inaddition to application in microbial screening, this method can be used toquantify glycolic acid. Four strains able to convert glycolonitrile to glycolicacid were isolated using this colorimetric method, among which N595displayed the highest hydrolytic activity. Strain N595 was identified asRhodococcus microorganism through a series of physiological andbiochemical tests. Substrate specificity suggested that Rhodococcus sp. N595 containedboth intracellular nitrile hydratase and amidase activities. Batch cultivationshowed that the optimal production medium consisted of 8 g/l mannitol, 4.9g/l peptone, 4.1 g/l yeast extract, 1 g/l K₂HPO₄, 1 g/l KH₂PO₄, 0.2 g/lMgSO₄, 0.03 g/l FeSO₄, 1 g/l NaCl, 0.5 g/l caprolactam.Bioconversion conditions and biotransformation process fromglycolonitrile to glycolic acid by Rhodococcus sp. N595 resting cells wereinvestigated. The optimal reaction temperature and pH were 42℃and7.5-7.8 in phosphate buffer solution. Glycolonitrile-hydrolyzing activitywas stable and the half-life was 45.2h at 42℃. Mg²⁺ and Fe²⁺ significantlyenhanced cell activity, however, either high concentration substrate orproduct exhibited inhibitive effect. At the preferred glycolonitrileconcentration of 1.6 g/l, Rhocococcus sp. N595 could perform 3-batchbiotransformation continuously, and the accumulated glycolic acidconcentration was 4.7 g/l. Product inhibition could occur when glycolicacid concentration reached 2 g/l. Finally, reaction kinetics parameters ofresting cells were tested. The maximum reaction rate V_m, the apparentMichaelis-Menten constant K_m, the substrate inhibitive constant K_s were2.816 mmol l^-1 min^-1, 12.157 mmol/l, 53.78 mmol/l, respectively.