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Biochemistry (Mosc). 2003 Nov;68(11):1189-94. |
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Expression and refolding of tobacco anionic
peroxidase from E. coli inclusion bodies.
Hushpulian D.M., Savitski P.A., Rojkova A.M., Chubar T.A.,
Fechina V.A., Sakharov I.Y., Lagrimini L.M., Tishkov V.I.*,
Gazaryan I.G.
*Department of Chemical Enzymology, Faculty of Chemistry,
M.V.Lomonosov Moscow State University, Moscow, 119992, Russian
Federation. vit@enz.chem.msu.ru
Coding DNA of the tobacco anionic peroxidase gene was cloned in
pET40b vector. The problem of 11 arginine codons, rare in
procaryotes, in the tobacco peroxidase gene was solved using E.
coli BL21(DE3) Codon Plus strain. The expression level of the
tobacco apo-peroxidase in the above strain was approximately 40%
of the total E. coli protein. The tobacco peroxidase refolding was
optimized based on the earlier developed protocol for horseradish
peroxidase. The reactivation yield of recombinant tobacco enzyme
was about 7% with the specific activity of 1100-1200 U/mg towards
2,2;-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was
shown that the reaction of ABTS oxidation by hydrogen peroxide
catalyzed by recombinant tobacco peroxidase proceeds via the
ping-pong kinetic mechanism as for the native enzyme. In the
presence of calcium ions, the recombinant peroxidase exhibits a
2.5-fold decrease in the second order rate constant for hydrogen
peroxide and 1.5-fold decrease for ABTS. Thus, calcium ions have
an inhibitory effect on the recombinant enzyme like that observed
earlier for the native tobacco peroxidase. The data demonstrate
that the oligosaccharide part of the enzyme has no effect on the
kinetic properties and calcium inhibition of tobacco peroxidase.
PMID: 14640960
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