ИСТИНА

D-amino acid oxidase (DAAO, EC 1.4.3.3) is a FAD-containing flavoprotein which fulfills important physiological functions in living organisms. Moreover, DAAO is also of high practical interest. The enzyme is used in fine organic synthesis, biosensors and medical treatment. The most large-scale and perspective field of DAAO application is two-step biocatalytic conversion of Cephalosporin 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. A series of TvDAAO mutants with improved properties were obtained last few years. Amino acid changes in positions 54 and 108 resulted in mutant enzymes with improved catalytic efficiency with CephC. Mutations in cofactor-binding domain increased thermal stability and catalytic efficiency with many D-amino acids. In this work, we combined 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.