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A. A. Poloznikov, G. S. Zakharova, T. A. Chubar, D. M. Hushpulian, I. G. Gazaryan, V. I. Tishkov
Catalytic
properties and stability of recombinant tobacco peroxidase
with amino acid change Ile37Me
Abstract
The
Ile37Met substitution was generated in tobacco anionic peroxidase
(TOP) by site-directed mutagenesis to mimic the soybean peroxidase
(SBP), in which Met37 is responsible for the increased thermal stability. TOP
Ile37Met has been expressed in E. coli BL21(DE3) CodonPlus
and accumulated in inclusion bodies. The expression level of the constructed
enzyme was approximately 40% of the total E. coli protein. The enzyme
was reactivated into an active and soluble form via a refolding procedure that
was optimized based on the earlier developed protocol for wild-type TOP. The
substrate specificity, catalytic activity and thermostability
of TOP Ile37Met were investigated. It was shown that the introduction of
Ile37Met mutation does not increase the stability of the enzyme and on the
contrary, leads to a reduction of the catalytic properties of the enzyme.
Key words: tobacco peroxidase, mutagenesis, refolding, thermostability.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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