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D. L. Atroshenko, S. A. Zarubina, M. D. Shelomov, I. V. Golubev, S. S. Savin, V. I. Tishkov
Preparation and characterization of
multi-points mutants of the yeast d-amino acid oxidase with improved stavility
ans activity
Abstract
D-amino acid oxidase (DAAO) is a
FAD-containing oxidoreductase which stereospecifically oxidases D-amino acids
producing α-keto-acids, ammonium ion and hydrogen
peroxide. The most important application of DAAO is Cephalosporin C
oxidation in biocatalytic process of preparation of 7-amino cephalospranic
acid. Recently single-point mutants of DAAO from yeast Trigonopsis
variabilis (TvDAAO) with increasedthermal stability or (and) improved
activity with Cephalosporin C have been prepared in our laboratory. In
present wor some positive amino changes were combined to produce mutant TvDAAOs
with two and four amino changes (TvDAAO M1 and TvDAAO M2,
respectively). As result catalytic constants increased about 1,8 and 4-fold in
comparison with wild-type enzyme. Thermal stability of mutant TvDAAOs were
2-3-fold higher compared to wt-TvDAAO.
Key words: D-amino acid oxidase,
Cephalosporin C, site-directed mutagenesis, rational design, kinetic
properties, thermal stability.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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