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Isolation of enantioselective α-hydroxyacid dehydrogenases based on a high-throughput screening method.

Bioprocess and biosystems engineering (2012-05-09)
Ya-Ping Xue, Wei Wang, Ya-Jun Wang, Zhi-Qiang Liu, Yu-Guo Zheng, Yin-Chu Shen
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
2,4-Dinitrophenylhydrazine, reagent grade, 97%
Supelco
2,4-Dinitrophenylhydrazine hydrochloric acid solution, ~0.005 M in ethanol, for TLC derivatization