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Influence of substitution Met104Leu, Met156Leu, Met209Leu on oxidation stability, catalytic properties and thermal stabilityтезисы доклада
- Авторы: Atroshenko D.L., Golubev I.V., Osipova T.A., Savin S.S., Tishkov V.I.
- Сборник: Program and Abstract Book of International Conference "Biocatalysis-2015: Fundamentals and Applications", Moscow region, Avantel Club Istra
- Тезисы
- Год издания: 2015
- Место издания: Innovations and High Technologies MSU Ltd Moscow
- Первая страница: 100
- Последняя страница: 100
- Аннотация: Our laboratory carry out systematic investigations structure and function relationships of the enzymes with rational design. One of the enzymes is D-amino acid oxidase from the yeast Trigonopsis variabilis (TvDAAO). This enzyme catalyzes oxidation of D-amino acids into corresponding α-keto acids coupled with formation of hydrogen peroxide and ammonium. This enzyme plays an important physiological role and possesses wide practical application. One of the important properties for practical use of enzymes is operational stability. TvDAAO resistance against hydrogen peroxide is the most important point, because H2O2 is a product of the reaction catalyzed by the enzyme. Moreover, in one of the most large-scaled process using TvDAAO — preparation of 7-aminocephalosporanic acid, hydrogen peroxide is added to the reaction system for a quantitative reaction. The residues of the methionine and cysteine in enzymes are most susceptible to oxidation. Due to its small size hydrogen peroxide is able to penetrate into the protein globule and oxidize solvent inaccessible residue of Met and Cys. In our study we investigated the role of Met residues in TvDAAO in the chemical stability of the enzyme. Met residues in positions 104, 156 and 209 were replaced with Leu because of similarity to Met. Plasmids with mutations in tvdaao gene were obtained by site-directed mutagenesis. Mutant TvDAAOs were expressed in E. coli cells. Mutant enzymes were obtained in high-purified form and were characterized. Influence of Met104Leu, Met156Leu and Met209Leu on oxidation stability, catalytic properties and thermal stability of TvDAAO will be discussed. The work is supported by Russian Foundation for Basic Research (grants #14-04-00865-а and 14-04-31194_mol-a).
- Добавил в систему: Тишков Владимир Иванович