ИСТИНА

D-amino acid oxidase is a FAD-containing enzyme that catalyzes oxidation of D-amino acids to corresponding α-keto acids. DAAO plays important role in living organisms and it is very attractive enzyme for pharmaceutical industry, fine organic synthesis and analytical biotechnology. DAAO is very important for biocatalytic production of wide range of optically pure compounds. D-amino acids oxidase from the yeast Trigonopsis variabilis (TvDAAO) is one of the most interesting enzyme in terms of biotechnological application due to high thermal stability and high catalytic activity towards many substrates. However, TvDAAO does not catalyze oxidation of some substrates like D-Lys or D-Glu due to the active site features. In our recent work the role of Met104 was studied with rational protein design [1]. The important role of Met104 in thermal stability and catalytic activity with D-amino acids, controlling access to the active site was shown. The aim of this research was the studying of the influence of Met104 negatively and positively charged substitutions. Site-directed mutagenesis of Met104 was performed to introduce Glu and Lys in 104th position. Both mutants were expressed in E.coli and obtained in soluble and active form. Two mutants were less stable than the wild type enzyme in the same extent. The outstanding result was the 17 times higher activity of TvDAAO Met104Glu on D-lysine in comparison to wild type, while TvDAAO Met104Lys was absolutely inactive on D-lysine as well as on D-Glu. Mutant TvDAAO Met104Glu could be used in selective biosensors for D-lysine detection and for biocatalytic oxidation of this substrate.