Optimization of HPLC technique for determining catalytic parameters of D-amino acid oxidase on cephalosporin C

Abstract

Approximately half of cephalosporin antibiotics of different generations are produced from 7-aminocephalosporanic acid, which to date is prepared by organic synthesis. Instead of organic synthesis, a two-step enzymatic process is gradually being developed. The first step is enzymatic oxidation of natural antibiotic cephalosporin C by D-amino acid oxidase (DAAO). Yeast enzymes are used for this purpose due to the highest activity on cephalosporin C. The standard technique of determining the activity of D-amino acid oxidase is based on determining the concentration of released hydrogen peroxide using horseradish peroxidase. During cephalosporin C oxidation, hydrogen peroxide is involved in the spontaneous nonenzymatic reaction with the intermediate product. Thus, monitoring the substrate consumption with high-performance liquid chromatography (HPLC) is the most correct way to determine the activity. In this paper, we have optimized the HPLC technique of determining the cephalosporin C concentration during its oxidation with D-amino acid oxidase in the reaction mixture. Using the optimized technique, we have determined the catalytic parameters for wild-type and mutant D-amino acid oxidase on cephalosporin C.