Preparation and Characterization of Multi-Points Mutants of the Yeast D-Amino Acid Oxidase with Improved Stavility and Activity
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.