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D. L. Atroshenko, A. A. Pometun, S. S. Savin, V. I. Tishkov
Determination of kinetic parameters of
wild-type and mutant D-amino acid oxidase from yeast in reaction of
cephalosporine C oxidation
Abstract
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.
Key words: D-amino acid oxidase, Trigonopsis
variabilis, recombinant enzyme, mutant forms, Cephalosporine C
oxidation, kinetic parameters.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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