ИСТИНА

D-amino acid oxidase is a FAD-containing enzyme that catalyzes oxidation of D-amino acids oxidation to corresponding α-keto acids. DAAO plays important role in regulation of many processes in living beings 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 and especially in synthesis of cephalosporin antibiotics. D-amino acids oxidase from yeast Trigonopsis variabilis (TvDAAO) is one of the most interesting enzyme in terms of biotechnological application due to thermal stability and catalytic activity to many substrates including Cephalosporin C. In our laboratory rational protein design technique was applied for optimizing properties of TvDAAO. In our recent work substitutions of Met104 with hydrophobic residues were studied with site-directed mutagenesis [1]. It was shown that Met104 plays important role in thermal stability and catalytic activity with D-amino acids, controlling access to the active site. The aim of this research was to study the influence of substitution of Met104 with negatively and positively charged amino acids. 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. It was shown that Met104Glu and Met104Lys produce negative influence on thermal stability of the enzyme in the same extent. Substrate specificity was also changed. It should be noted that catalytic activity of Met104Glu was a 17 times higher in comparison to wild type.