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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.
Moscow University Chemistry Bulletin.
2014, Vol. 55, No. 2, P. 106
   

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