Activity of yeast D-amino acid oxidase on aromatic unnatural amino acids

D-Amino acid oxidase is a FAD-dependent enzyme that catalyses the conversion of the D-enantiomer of amino acids into the corresponding a-keto acid. Substrate specificity of the enzyme from the yeast Rhodotorula gracilis was investigated towards aromatic amino acids, and particularly synthetic a-amino acids. A significant improvement of the activity (max,app)) and of the specificity constant (the V-max,V-app/K-m,K-app ratio) on a number of the substrates tested was obtained using a single-point mutant enzyme designed by a rational approach. With R. gracilis D-amino acid oxidase the complete resolution Of D,L-homo-phenylalanine was obtained with the aim to produce the corresponding pure L-isomer and to use the corresponding a-keto acid as a precursor of the amino acid in the L-form.