ИСТИНА

D-amino acid oxidase (DAAO) plays important role in living systems. The enzyme from yeast Trigonospsis variabilis (TvDAAO) is also of high practical importance. It is used in biocatalytic two-enzyme process of preparation of 7-amino cephalosporanic acid from cephalosporin C. The main drawback of TvDAAO is insufficient thermal and chemical stability. Using high speed sedimentation analysis and light scattering technique it was shown that oligomeric state of TvDAAO depends on concentration. At concentrations 5-30 µg/ml the enzyme is a homodimer. At concentration higher than 50 µg/ml oligomeric state became tetrameric and higher. At concentrations lower than 0.5 µg/ml dimer TvDAAO dissociates to subunits. Stability of dimer can be improved by addition of FAD. Analysis of TvDAAO activity at different concentrations showed that only dimer is active. Hydrogen peroxide is the product of TvDAAO reaction. It can inactivate the enzyme through oxidation of Met and Cys residues. Site-directed mutagenesis of four non-conservative Met residues showed that these residues do not control the TvDAAO chemical stability but substitution Met156Leu increased thermal stability. Cys108 is placed in D-amino acid binding domain. Site directed mutagenesis of Cys108 for Ala and Ser residues improved the chemical but not thermal stability. Substitution Cys108Phe increased both chemical and thermal stability. Amino acid changes Cys108Phe and Cys108Ala resulted in increase of catalytic efficiency with cephalosporin C by 3 and 4-fold, respectively. The second Cys residue in position 298 is placed in oxygen channel of active site. It was changed with four residues. Amino acid changes Cys108Phe and Met156Leu were combined with substitution Phe54Ser to produce active with cephalosporin C stable mutant enzyme with increased thermal and chemical stability. This work was supported by Russian Science Foundation (grant 16-14-00043). Опубликовано в FEBS Journal, 2017, т.284, №S1, p.199-200