ИСТИНА

D-amino acid oxidase (DAAO) is a well known FAD-containing enzyme catalyzing oxygen-dependent D-amino acids oxidation to α-keto acids with absolute stereospecificity. DAAO plays important role in regulation of many processes in living beings. DAAO is the enzyme of high practical interest for pharmaceutical industry, analytical biotechnology (for detection of D-amino acids) and fine organic synthesis of unnatural L-amino acids and α keto acids at nowadays. DAAO is the most widely used for biocatalytic conversion of cephalosporin C into 7-amino cephalosporanic acid (7-ACA) being the key precursor for synthesis of cephalosporanic antibiotics. D-amino acids oxidase from the yeast Trigonopsis variabilis (TvDAAO) is the most promising enzyme for application due to the broad substrate specificity and high thermal stability among studied DAAOs. Nevertheless wild type TvDAAO often possesses non-optimal properties for practical processes. Properties of the enzyme can be optimized by rational protein design. TvDAAO has been studied with rational protein design for a several years in our laboratory. During this time new mutant TvDAAOs with changed substrate specificity and higher thermal stability have been obtained. However study of structure functional relationships is still of high practical and theoretical interest. We will present the latest results obtained in our laboratory with rational design. This technique includes computer analysis of TvDAAO’s structure, docking of substrates to the active site, alignment of DAAOs sequences and simulation of mutations. The most perspective substitutions in the active site were chosen by the screening mentioned above. As the result new mutant TvDAAOs with modified properties were obtained. Influence on catalytic properties and thermal stability of introduced mutations and possibility of biotechnological application of new mutant enzymes will be discussed. The work is supported by Russian Foundation for Basic Research (grant #11-04-00959-а) and grant “U.M.N.I.K.” (I.V. Golubev) from the Russian Foundation for Assistance to Small Innovative Enterprises (FASIE).