ИСТИНА

D-amino acid oxidase (DAAO, EC 1.4.3.3) is a FAD-containing enzyme which fulfills physi-ological functions in living organisms. Also DAAO is the important biotechnological enzyme being used in biosensors and fine organic synthesis, especially in two step enzymatic conversion of cepha-losporin C (CephC) into 7-amino cephalosporanic acid. In our laboratory gene of DAAO from yeast Trigonopsis variabilis (TvDAAO) was cloned and expressed in E.coli. Amino acid changes in positions 54 and 108 resulted in mutant enzymes with improved catalytic efficiency with CephC. Some mutations in cofactor-binding domain increased thermal stability catalytic efficiency with many D-amino acids. Here we present the results of combining these amino acid changes in multi-point mutants. The plasmids with mutations in tvdaao gene providing triple and quaternary amino acid substitutions were obtained. Mutant enzymes were expressed in E.coli cells, purified and characterized. It was found that new multi-point mutants of TvDAAO showed further enhancing in catalytic activity and thermal stability. Chromatographic activity determination with CephC was performed. The work was supported by Russian Foundation for Basic Research (grants 14-04-00865-a, 14-04-32064-mol_a)