Preparation and characterization of multipoint yeast D-amino acid oxidase mutants with improved stability and activity

D-amino acid oxidase (DAAO) is an FAD-containing oxidoreductase that stereospecifically oxidases D-amino acids to produce α-keto-acids, an ammonium ion, and hydrogen peroxide. The most important biotechnological process involving DAAO is the production of 7-amino cephalospranic acid (7-ACA) from cephalosporin C. The reaction product, 7-ACA, is then used as a precursor for the synthesis of cephalosporin antibiotics of different generations. We previously obtained mutant DAAOs from the yeast Trigonopsis variabilis (TvDAAO). The mutants with point amino acid substitutions were characterized by either an increased thermal stability or improved catalytic properties in the oxidation of cephalosporin C. In the present study, we obtained two new mutant TvDAAOs with two and four amino acid substitutions, respectively. The catalytic constants of these mutant TvDAAOs for the oxidation of cephalosporin C were 1.8 and 4 times higher than the respective parameter of the wild-type enzyme (wt-TvDAAO). The combination of substitutions increased the thermal stabilities of both mutant TvDAAOs by a factor of 2–3 as compared with the wt- TvDAAO.