Rational design of D-amino acid oxidase to improve enzyme properties

Zarubina, S.A.; Golubev, I.V.; Tishkov, V.I.

Авторы: Zarubina S.A., Golubev I.V., Tishkov V.I.
Сборник: Book of abstracts of IX International conference of young scientists on chemistry “Mendeleev - 2015”
Тезисы
Год издания: 2015
Место издания: Saint Petersburg State University St.-Petersburg
Первая страница: 333
Последняя страница: 333
Аннотация: D-amino acid oxidase (DAAO) is a FAD-containing enzyme which plays essential physiological functions in living organisms. The enzyme is used in fine organic synthesis, biosensors and medical treatment. Moreover, the most large-scale and perspective field of DAAO application is made use of cephalosporin antibiotics production. There is two-step biocatalytic conversion of Cephalosporin C (CephC) into 7-aminocephalosporanic acid. In our laboratory the rational design is used to obtain mutant forms of DAAO from yeast Trigonopsis variabilis (TvDAAO). Positions were determined by simulating mutant structures. Amino acid changes in active site resulted in mutant enzymes with enhanced catalytic efficiency with CephC. Mutations in cofactor-binding domain increased catalytic efficiency with many D-amino acids and thermal stability. In current work, we combined these amino acid changes into 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. Chromatographic activity determination with CephC was performed. It was found that new multi-point mutants of TvDAAO showed enhanced catalytic activity and thermal stability compared to initial enzymes. This work was supported by Russian Foundation for Basic Research (grants 14-04-00865-a, 14-04-32064-mol_a and 14-04-31194-mol_a).
Добавил в систему: Тишков Владимир Иванович

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