Analyzing the d-amino acid content in biological samples by engineered enzymes

The aim of our present research is to produce mutant forms of d-amino acid oxidase from Rhodotorula gracilis in order to determine d-amino acid content in different biological samples. During the past few years, our group has produced yeast d-amino acid oxidase variants with altered substrate specificity (e.g., active on acidic, or hydrophobic, or on all d-amino acids) both by rational design and directed evolution methods. Now, the kinetic constants for a number of amino acids (even for unnatural ones) of the most relevant d-amino acid oxidase variants have been investigated. This information constitutes the basis for considering potential analytical applications in this important field.