Online Enzyme Discrimination And Determination Of Substrate Enantiomers Based On Electrophoretically Mediated Microanalysis

Enzyme is an important biocatalyst in many areas of life, medicine and food. Electrophoresis media microanalysis (EMMA) technology has unique advantages in the study of enzyme analysis and attracted widespread attention. The purpose of this paper is to develop fast, simple, accurate online discrimination and determination of substrate enantiomers with the enzyme chiral characteristics. In this work, we proposed the first application of an electrophoretically mediated microanalysis (EMMA) method, toghther with characteristics of chiral enzyme for fast online discrimination and determination of substrate enantiomers, which was successfully used to determine the L-/D-lactate in several yogurt and wine samples.In this paper, we utilized the high stereoselectivity of enzymes and the different electrophoretic migration velocity between enzymes and samples, and carried out a modified EMMA mode of successively injecting enzyme enantiomers and samples into the capillary. We optimized the experimental conditions, including EMMA injection conditions, the buffer solution and the coenzyme NAD+and the enzyme LDH concentration. In the optimiz ed the experimental conditions, the chiral discrimination can be achieved with high accuracy in less than2min and resolution larger than11.0. Excellent linear dependence of the two NADH peak intensities on the concentration of the corresponding lactate enantiomers was obtained (R2>0.99). Good repeatability of online chiral discrimination was demonstrated with relative standard deviation (RSD)<1.5%for migration time (n=5). The limit of detection was26μ M for D-lactate and49μ M for L-lactate (S/N=3).Km values for L-and D-lactate were measured and were consistent with those of the off-line enzyme assays,confirming the accuracy of the method. Lactic acid contents in the three commercially available yogurt and two red wine were determined, demonstrating the utility of the method. In conclution, The method that takes advantage of the characteristics of chiral enzymes instead of the choice of a suitable chiral selector presents fast online discrimination and determination of substrate enantiomers, which was achieved by just one EMMA assay. And the method extends the application of biological enzymes and EMMA technology.