ИСТИНА

D-amino acids oxidase (DAAO) is the enzyme of high practical interest. DAAO is used in analytical biotechnology for detection of D-amino acids in different samples, in fine organic synthesis of unnatural L-amino acids and α keto acids. The most large scale process involving DAAO is the two step enzymatic conversion of cephalosporin C into 7-amino cephalosporanic acid (7-ACA). 7-ACA is the key precursor for synthesis of cephalosporanic antibiotics. D-amino acids oxidase from the yeast Trigonopsis variabilis (TvDAAO) possesses the best properties among all known DAAO’s. Nevertheless properties of wild type enzyme are very often non-optimal for practical processes. Properties of enzyme can be improved by protein design technique. Here we will present the results of protein engineering of TvDAAO. Computer analysis of TvDAAO and docking of substrates to active site of the enzyme were performed, amino acid residues controlling access to the active site were identified. The most appropriate substitutions were proposed based on virtual screening of potential mutants. New mutant TvDAAO’s with changed substrate specificity (more narrow spectrum) were obtained. Catalytic activity with some substrates including cephalosporin C as well as thermal stability were improved. Possibility of application of new mutant TvDAAO’s for early detection of neurodegenerative disorders related to D amino acids derangements was considered. The work is supported by Russian Foundation for Basic Research (grant #11-04-00959-а).