title: Online enzyme discrimination and determination of substrate enantiomers based on electrophoretically mediated microanalysis.
abstract: We proposed the first application of an electrophoretically mediated microanalysis (EMMA) method for fast online discrimination and determination of substrate enantiomers, which was achieved by just one EMMA assay. Lactate dehydrogenase (LDH)-catalyzed reaction was studied to evaluate the feasibility and performance of the presented method. The L- and D-LDH chiral enzymatic reactions, which are highly stereoselective to the lactate enantiomers, were initiated successively in one capillary, and the corresponding products, nicotinamide adenine dinucleotide (NADH), were online discriminated and detected by UV absorption. Excellent linear dependence of the two NADH peak intensities on the concentration of the corresponding lactate enantiomers was obtained within a wide range of 0.1-10 mM. The limit of detection was 26 μM for D-lactate and 49 μM for L-lactate (S/N = 3). Good repeatability of online chiral discrimination was demonstrated with relative standard deviation (RSD) < 6.3% for NADH peak height and RSD < 1.5% for migration time (n = 5). K(m) values for L- and D-lactate were measured and were consistent with those of the off-line enzyme assays. The presented method was successfully applied to determine the L-/D-lactate in several yogurt and wine samples. Our study shows a new application of the EMMA method utilizing high stereoselectivity of enzymes for fast online chiral analysis.
authors: Faculty of Chemistry, Northeast Normal University, Changchun, Jilin, 130024, People's Republic of China.
authors: Wenwen
authors: W
authors: Zhao
authors: Miaomiao
authors: M
authors: Tian
authors: Rongbin
authors: R
authors: Nie
authors: Yulin
authors: Y
authors: Wang
authors: Liping
authors: L
authors: Guo
authors: Li
authors: L
authors: Yang
mesh_terms: D019075:Electrophoresis, Capillary; D057075:Enzyme Assays; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D045022:Lactate Dehydrogenases / Q000378:metabolism; D019344:Lactic Acid / Q000737:chemistry / Q000378:metabolism; D009243:NAD / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D013379:Substrate Specificity; D014920:Wine / Q000032:analysis; D015014:Yogurt / Q000032:analysis
chemical_list: D009243:NAD; D019344:Lactic Acid; D045022:Lactate Dehydrogenases; D007770:L-Lactate Dehydrogenase; C071195:D-lactate dehydrogenase