Determination of the Kinetic Parameters of a Wild-Type D-Amino Acid Oxidase from Yeast and Its Mutant Forms in a Reaction of Cephalosporin C Oxidation

Аннотация

The oxidation of cephalosporin C (CPC) by D-amino acid oxidase is the first stage in the biocatalytic industrial process of the production of 7-aminocephalosporanic acid, the initial synthon for the production of semisynthetic cephalosporin antibiotics. We determined the kinetic parameters (catalytic constant rate and Michaelis constant) of the wild-type recombinant oxidase from the yeast Trigonopsis variabilis (TvDAAO); three mutant forms of the enzyme with point Met-to-Leu substitutions at positions 104, 156, and 209; and the TvDAAO RDAF quadruple mutant using the dependence of the initial reaction rate on the CPC concentration (the initial rate method). The point substitutions in the mutants are shown to lead primarily to a decrease in the Michaelis constant. The largest (39%) increase in catalytic efficiency k_cat/K_M is observed for the mutant with the Met156Leu substitution. The Michaelis constant for the increased four times compared to that for the wild-type enzyme in the reaction with CPC, whereas the catalytic efficiency remains almost unchanged due to a four-fold increase in the catalytic rate constant.