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