To isolate enantioselective α-hydroxyacid dehydrogenases (α-HADHs), a high-throughput screening method was established. 2,4-Dinitrophenylhydrazine solution forms a red-brown complex with ketoacid produced during the α-HADH-mediated oxidation of α-hydroxyacid. The complex can be easily quantified by spectrophotometric measurement at 458 nm. The enantioselectivity of α-HADH in each strain can be measured with this colorimetric method using (R)- and (S)-α-hydroxyacid concurrently as substrates to evaluate the apparent enantioselectivity (E app). The E app closely matches the value of true enantioselectivity (E true) determined by HPLC analysis. With this method, a total of 34 stains harboring enantioselective α-HADHs were selected from 526 potential α-HADH-producing microorganisms. Pseudomonas aeruginosa displayed the highest (S)-enantioselective α-HADH activity. This strain appears promising for potential application in industry to produce (R)-α-hydroxyacids. The method described herein represents a useful tool for the high-throughput isolation of enantioselective α-HADHs.
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This work was supported by the Fund of the National High Technology Research and Development Program of China (863 Program) (No. 2011AA02A210), the Major Basic Research Development Program of China (973 Project) (No. 2009CB724704) and Natural Science Foundation of Zhejiang Province (No. Z4090612 and No. Y4080334).
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Xue, Y., Wang, W., Wang, Y. et al. Isolation of enantioselective α-hydroxyacid dehydrogenases based on a high-throughput screening method. Bioprocess Biosyst Eng 35, 1515–1522 (2012). https://doi.org/10.1007/s00449-012-0741-1
- High-throughput screening
- α-Hydroxyacid dehydrogenase
- Pseudomonas aeruginosa