A chiral ligand exchange CE essay with zinc(II)–l-valine complex for determining enzyme kinetic constant of l-amino acid oxidase

A new strategy for the enantioseparation of d,l-amino acids employing the principle of ligand exchange capillary electrophoresis with Zn(II)–l-valine complex as a chiral selecting system in the presence of β-cyclodextrin has been designed. Successful enantioseparation of label free and labeled amino acids have been achieved with a buffer of 100.0 mM boric acid, 5.0 mM ammonium acetate, 4.0 mM β-cyclodextrin, 4.0 mM ZnSO4 and 8.0 mM l-valine at pH 8.1. This new method was shown to be applicable to the quantitative analysis of label free d- and l-aromatic amino acids. Furthermore, the expanding enzymatic use of l-amino acid oxidase to incubate with different l-amino acids has allowed understanding of the substrateʹs specificity. An on-column incubation assay has been developed to study the l-amino acid oxidaseʹs catalytic efficiency. It was demonstrated that the enzyme kinetic constant could be determined by using this new method.