title: Isolation of enantioselective α-hydroxyacid dehydrogenases based on a high-throughput screening method.
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.
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Xue
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Ya-Jun
authors.initials: YJ
authors.lastname: Wang
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Liu
authors.forename: Yu-Guo
authors.initials: YG
authors.lastname: Zheng
authors.forename: Yin-Chu
authors.initials: YC
authors.lastname: Shen
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D010659:Phenylhydrazines / Q000737:chemistry*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D020579:Sinorhizobium / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D010659:Phenylhydrazines; C004787:2,4-dinitrophenylhydrazine; D000429:Alcohol Oxidoreductases; C021379:D-2-hydroxyacid dehydrogenase