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D-amino acid oxidase (DAAO) is FAD-containing enzyme that found wide application in fine organic synthesis and analytical biotechnology due to the ability of oxidation D-amino acids with absolute stereo specificity. The most prominent process involving DAAO is the two-step enzymatic conversion of Cephalosporin C (CephC) into 7-amino cephalosporanic acid (7-ACA). D-amino acid oxidase from Trigonopsis variabilis (TvDAAO) possesses the best properties for biotechnological application. However wild type enzyme doesn’t entirely fit practical requirements in terms of thermal stability and catalytic activity. But TvDAAO properties can be optimized with rational protein design. Rational design approach was applied for producing mutant TvDAAOs with improved thermal stability and higher catalytic activity to CephC. CephC was docked in the enzyme active site and amino acid residues interacting with substrate molecule were identified. Site directed mutagenesis of residues concerned in some cases resulted in 2-3 times higher catalytic activity with CephC and other D-amino acids or higher thermal stability. Further combination of positive amino changes in one mutant enzyme produced biocatalysts with higher catalytic efficiency and thermal stability. New mutant TvDAAOs may be valuable for biotechnological production of 7-ACA, chiral synthesis and biosensors as well.