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I. V. Golubev, N. V. Komarova, O. E. Skirgello, T. A. Osipova, V. I. Tishkov

Optimization of HPLC technique of determination of catalytic parameters of D-amino acid oxidase in reaction of cephalosporin C oxidation

Abstract

Half of the amount of cephalosporin antibiotics of different generations is produced from 7-aminocephalosporanic acid, which is prepared with organic synthesis so far. In place of the organic synthesis is gradually coming two-step enzymatic process. The first step is the enzymatic oxidation of natural antibiotic cephalosporin C  with D-amino acid oxidase. Yeast enzymes are used for this purpose due to the highest activity towards cephalosporin C. The standard technique of determining of the activity of D-amino acid oxidase is based on determination of the concentration of released hydrogen peroxide using horseradish peroxidase. During cephalosporin C oxidation hydrogen peroxide is involved in spontaneous non-enzymatic reaction with intermediate product. So the most correct for the activity determination is to monitor for the substrate consumption with high performance liquid chromatography. In this paper we have optimized HPLC technique of determination of cephalosporin C concentration during its oxidation with D-amino acid oxidase in the reaction mixture. Using optimized technique we have determined catalytic parameters for wild type and mutant D-amino acid oxidase with cephalosporin C.
Key Words: D-amino acid oxidase, Trigonopsis variabilis, Cephalosporin C, catalytic parameters, high performance liquid chromatography.
Moscow University Chemistry Bulletin.
2014, Vol. 55, No. 2, P. 93
   

Copyright (C) Chemistry Dept., Moscow State University, 2002
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