DETERMINATION OF KINETIC PARAMETERS OF WILD-TYPE AND MUTANT D-AMINO ACID OXIDASE FROM YEAST IN REACTION OF CEPHALOSPORINE C OXIDATION
Cephalosporin C (CPC) oxidation by D-amino acid oxidase (DAAO) is the first reaction in industrial biocatalytic process of production of 7-aminocephalosporanic acid which is used as synthon for preparation of different semi-synthetic cephalosporin antibiotics. Using “initial rates” approach we determined kinetic parameters - efficient catalytic constant and Michaelis constant for CPC, for wild-type and mutant DAAO from yeast Trigonopsis variabilis (TvDAAO). Three single-point mutants with changes Met/Leu in positions 104, 156 and 209, as well as for four-points mutant TvDAAO RDAF were studied. It was shown that substitutions Met/Leu resulted in slight decrease of KM. Maximal increase of catalytic efficiency kcat/KM 39% was observed for mutant with amino acid change Met156Leu. In the case of mutant TvDAAO RDAF catalytic efficiency is the same as for wild-type enzyme because both parameters, kcat and KM increased simultaneously four times.