ИСТИНА

D-amino acid oxidase from the yeast Trigonopsis variabilis (TvDAAO) is widely used for fine organic synthesis and in bi-enzyme process of preparation 7-aminocephalosporanic acid from Cephalosporin C. Hydrogen peroxide is the product of reactions catalyzed by TvDAAO. Oxidation of the enzyme by H2O2 is one of the main reasons of TvDAAO inactivation during process. In previous work we carried out site directed mutagenesis to study of role of Cys108 and Cys294 in catalysis and stability of TvDAAO. In present work we checked role of some Met residues in catalysis, chemical and thermal stability of the enzyme. Method to study inactivation of wild-type and mutant TvDAAOs in presence of hydrogen peroxide was developed. Analysis of TvDAAO structure resulted in selection of three Met residues which are not conservative and which could be accessible to solvent. Three single-point mutant TvDAAO with changes Met104Ile, Met156Leu, and Met209Leu were prepared, expressed in E.coli and purified. Catalytic properties with different D-amino acids as well as thermal and chemical stability were studied. It was shown that all mutation did not change chemical stability, while one mutant had higher thermal stability. Catalytic parameters of wild-type and mutant TvDAAOs in reaction of oxidation of Cephalosporin C were determined with HPLC method using steady-state and integral kinetics. It was found that some amino changes improve catalytic efficiency. This work was supported by grant of Russian Science Foundation (grant 16-04-00043.