ИСТИНА

D-amino acid oxidase (DAAO, EC 1.4.3.3) is FAD containing enzyme and catalyses oxidative deamination of D-amino acids to corresponding α ketoacids. DAAO from yeasts Trigonopsis variabilis (TvDAAO) shows the highest thermal stability and activity with cephalosporin C. The enzyme is the best catalyst among known DAAOs for use in two step biocatalytic process of production of 7-aminocephalosporanic acid – the key compound for obtaining semi-synthetic cephalosporin antibiotics. It is also used for preparation of unnatural L-amino acids and α ketoacids. For efficient practical application of TvDAAO the enzyme stability as well as profile of substrate specificity have to be improved. Previously we cloned and expressed gene of TvDAAO in E.coli cells. Crystals of mutant TvDAAO were prepared and structure was solved with resolution 1.8 Å. Analysis of the enzyme 3D structure revealed potential sites for directed mutagenesis to improve thermal stability and change substrate specificity. More than 20 mutant TvDAAOs were prepared and characterized. It was shown that amino acid substitutions resulted in significant change of thermal stability and provide mutant TvDAAO with different substrate specificity profile. Use for mutant TvDAAOs for fine organic synthesis as well in medicine diagnostics is discussed. This work was supported by Russian Foundation for Basic Research (grant 11-04-00959) and Ministry of Education and Sciences of Russian Federation (contract 16.512.11.2253)