Abstract
Tobacco anionic peroxidase (TOP) mutant Ile37Met was produced by site-directed mutagenesis to mimic soybean peroxidase (SBP), in which Met37 is responsible for increased thermal stability. TOP Ile37Met was expressed in E. coli BL21(DE3) CodonPlus in the form of 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 earlier developed for wild-type TOP. The substrate specificity, catalytic activity, and thermal stability of TOP Ile37Met were investigated. It was shown that the introduction of the Ile37Met mutation does not increases the stability of the enzyme; on the contrary, it leads to a reduction in the catalytic properties of the enzyme.
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Gazarian, I.G., Lagrimini, L.M., George, S.J., and Thorneley, R.N.F., J. Biochem., 1996, vol. 320, p. 369.
Gazaryan, I.G., Rubtsova, M.Yu., Kapeliuch, Yu.L., Rodriguez-Lopez, J.N., Lagrimini, L.M., and Thorneley, R.N.F., Photochem. Photobiol., 1998, vol. 67, p. 106.
Gazaryan, I.G., and Lagrimini, L.M., Phytochemistry, 1996, vol. 41, p. 1029.
McEldoon, J.P., and Dordick, J.S., Biotechnol. Progr., 1996, vol. 12, p. 555.
McEldoon, J.P., Pokora, A.R., and Dordick, J.S., Enzyme Microb. Technol., 1995, vol. 17, p. 359.
Henriksen, A., Mirza, O., Indiani, C., Teilum, K., Smulevich, G., Welinder, K.G., and Gajhede, M., Protein Sci., 2001, vol. 10, p. 108.
Amisha, K.J.K. and Digambar, V.B., Biochem. Eng. J., 2008, vol. 38, p. 110.
Hushpulian, D.M., Savitski, P.A., Rojkova, A.M., Chubar, T.A., Fechina, V.A., Sakharov, I.Yu., Lagrimini, L.M., Tishkov, V.I., and Gazaryan, I.G., Biochemistry (Moscow), 2003, vol. 68, no 11, p. 1189.
Patterson, W.R. and Poulos, T.L., Biochemistry, 1995, vol. 34, p. 4331.
Piontek, K., Glumoff, T., and Winterhalter, K., FEBS Lett., 1993, vol. 315, p. 119.
Laemmli, U.K., Nature, 1970, vol. 227(5259), p. 680.
Childs, R.E. and Bardsley, W.G., Biochem. J., 1975, vol. 145, p. 93.
Hosoya, T., J. Biochem., 1960, vol. 47, p. 794.
Gallati, H., J. Clin. Chem. Clin. Biochem., 1997, vol. 15, p. 699.
Hasinoff, F. and Dunford, H.B., Biochemistry, 1970, vol. 9, p. 4930.
Lebedeva, O.V., Ugarova, N.N., and Berezin, I.V., Biokhimiya (Moscow), 1977, vol. 42, p. 1372.
Gallati, H. and Brodbeck, H., J. Clin. Chem. Clin. Biochem., 1982, vol. 20, p. 221.
Paler-Martinez, A., Panus, P.C., Chumley, P.H., Ryan, U., Hardy, M.M., and Freeman, B.A., Arch. Biochem. Biophys., 1994, vol. 311, p. 79.
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Original Russian Text © A.A. Poloznikov, G.S. Zakharova, T.A. Chubar, D.M. Hushpulian, I.G. Gazaryan, V.I. Tishkov, 2014, published in Vestnik Moskovskogo Universiteta. Khimiya, 2014, No. 2, pp. 106–112.
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Poloznikov, A.A., Zakharova, G.S., Chubar, T.A. et al. Catalytic properties and stability of recombinant tobacco peroxidase with Ile37Met substitution. Moscow Univ. Chem. Bull. 69, 80–85 (2014). https://doi.org/10.3103/S0027131414020072
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DOI: https://doi.org/10.3103/S0027131414020072