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A. A. Poloznikov, P. A. Savitsky, D. M. Khushpulyan, T. A. Chubar, I. G. Gazaryan, V. I. Tishkov
Preparation and properties of
mutant tobacco peroxidase with additional tryptophan residues
Abstract
Mutant forms of tobacco
peroxidase Gln116Trp and Leu157Trp have been prepared and expressed in E.
coli cells BL21(DE3) CodonPlus. The muteins were expressed at the level up
to 40% of total E.coli proteins as inclusion bodies. Both mutants were prepared
inactive form according to standard refolding procedure optimized for wild-type
TOP. Yield of active proteins was about 9% and specific activity with ABTS –
2000–3000 U per mf of protein. Kinetoc properties of mutant enzymes in reaction
of ABTS oxidation with hydrogenperoxide have been studied. It was shown that
reaction proceeds through ping-pong kinetic mechanism but rate constants for
separate stages were different for mutants. It was supposed that additional
tryptophane residues participate in enzyme catalysis.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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