• R. Pliego-Arreaga
  • C. Regalado
  • A. Amaro-Reyes
  • B.E. García-Almendárez
Keywords: Brassica napus, peroxidase, expression, lactose, fermenter


Turnip (Brassica napus L. var. purple top white globe) peroxidases (E.C. may be applied free or immobilized on phenolic compounds degradation in waste water, in addition to its use in diagnostic test kits. The aim of this work was to achieve heterologous expression by lactose-induction of one peroxidase isoform (PodC) using Escherichia coli Rosetta 2 to produce recombinant turnip peroxidase in a fermenter. Recombinant PodC contained in inclusion bodies was refolded under oxidative conditions and purified by IMAC with a yield of 36 mg L-1 of culture. The enzyme showed a specific activity of 1004 ABTS units mg-1 under optimum conditions (pH 6, 45 ◦C). The value of KM for ABTS was 0.38 mM and the catalytic efficiency (kcat=KM) was 5.4×106 s-1 M-1. The use of lactose as inducer and scale up of the fermentation process may effectively decrease the production cost of recombinant PodC, which in turn can be applied for biotechnological applications


Agostini, E., Hernandez-Ruíz, J., Arnao, M.B., Milrad, S.R., Tigier, H.A. and Acosta, M. (2002). A peroxidase isoenzyme secreted by turnip (Brassica napus) hairy-root cultures: inactivation by hydrogen peroxide and application in diagnostic kits. Biotechnology and Applied Biochemistry 35, 1-7.

Bradford, M.M.A. (1976). A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.

Childs, R.E. and Bardsley, W.G. (1975). The steady-state kinetics of peroxidase with 2,2-azinodi(3-ethylbenzthiazolin-6-sulphonic acid) as chromogen. Biochemistry Journal 145, 93-103.

Deutscher, J., Francke, C. and Postma, P.W (2006). How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria. Microbiology and Molecular Biology Research 70, 939-1031.

Duarte-Vázquez, M.A., Whitaker, J.R., Rojo-Domínguez, A., García-Almendárez, B.E. and Regalado, C. (2003). Isolation and thermal characterization of an acidic isoperoxidase from turnip roots. Journal of Agricultural and Food Chemistry 51, 5096-5102.

Gazaryan, I.G., Dosseva, V.V., Galkin, A.G. and Tishkov, V.I. (1994). Effect of single-point mutations Phe41!His and Phe143!Glu on folding and catalytic properties of recombinant horseradish peroxidase expressed in E. coli. FEBS Letters 354, 248-250.

Grigorenko, V., Andreeva, I., Bochers, T., Spener, F. and Egorov, A. (2001). A genetically engineered fusion protein with horseradish peroxidase as a marker enzyme for use in competitive immunoassays. Analytical Chemistry 73, 1134-1139.

Hernández-Martínez, R., Sancho-Solano, A., Loera Corral, O., Rojo-Domínguez, A., Regalado González, C., Huerta-Ochoa, S. and Prado-Barragan, L.A. (2011). Purification and characterization of a thermostable alkaline protease produced by Yarrowia lipolytica. Revista Mexicana de Ingeniería Química 10, 333-341.

Hushpulian, D.M., Savitski, P.A., Rojkova, A.M., Chubar, T.A., Fechina, V.A., Sakharov, I.Y., Lagrimini, L.M., Tishkoy, V.I., and Gazaryan, I.G. (2003). Expression and refolding of tobacco anionic peroxidase from E. coli inclusion bodies. Biochemistry (Moscow) 68, 1189-1194.

Ikonomou, L., Schneider, Y.J. and Agathos, S.N. (2003). Insect cell culture for industrial production of recombinant proteins. Applied Microbiology and Biotechnology 62, 1-20.

Lara, A.R. (2011). Recombinant protein production in Escherichia coli. Revista Mexicana de Ingeniería Química 10, 209-223.

Menzella, H.G., Ceccarelli, E.A. and Gramajo, H.C. (2003). Novel Escherichia coli strain allows efficient recombinant protein production using lactose as inducer. Biotechnology and Bioengineering 82, 809-817.

Pham, L.T.M., Kim, S.J., Song, B.K. and Kim, Y.H. (2011). Optimized refolding and characterization of S-peroxidase (CWPO C of Populus alba) expressed in E. coli. Protein Expression and Purification 80, 268-273.

Quintanilla-Guerrero, F., Duarte-Vázquez, M.A., Tinoco, R., Gómez-Suárez, M., García Almendárez, B.E., Vázquez-Duhalt, R. and Regalado, C. (2008). Chemical modification of turnip peroxidase with methoxypolyethylene glycol enhances activity and stability for phenol removal using the immobilized enzyme. Journal of Agricultural and Food Chemistry 56, 8058-8065.

Regalado, C., García-Almendarez, B.E. and Duarte-Vázquez, M.A. (2004). Biotechnological applications of peroxidases. Phytochemistry Reviews 3, 243-5669.

Rodríguez-Cabrera, N.A., Regalado, C. and Garc´ıa-Almendárez, B.E. (2011). Cloning,heterologous expression and properties of a recombinant active turnip peroxidase. Journal of Agricultural and Food Chemistry 59, 7120-7126.

Romero-Gómez, S., Duarte-Vázquez, M.A., García Almendárez, B.E., Mayorga-Martínez, L.,
Cervantes-Áviles, O. and Regalado, C. (2008). A putative peroxidase cDNA from turnip and analysis of the encoded protein sequence. Plant Foods for Human Nutrition 63, 157-162.

Ryan, B.J. and O'FagÁin, C. (2008) Effect of mutations in the helix Gregion of horseradish peroxidase. Biochimie 90, 1414-1421.

Sambrook, J. and Russell, D.W. (2001). Molecular cloning: A laboratory manual, 3rd ed. Cold Spring Harbor Lab Press New York 1.116-1.118.

Segura, M.M., Levin, G., Miranda, M.V., Mendive, F.M., Targovnik, H.M. and Cascone, O. (2005). High-level expression and purification of recombinant horseradish peroxidase isoenzyme C in SF-9 insect cell culture. Process Biochemistry 40, 795-800.

Smith, A.T., Santama, N., Dacevs, S., Edwards, M., Bray, R.C., Thomeleyn, R.N.F. and Burke, J.F. (1990). Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with Ca2+ and heme. Journal of Biological Chemistry 265, 13335-13343.

Studier, F.W. (2005). Protein production by autoinduction in high density shaking cultures. Protein Expression and Purification 41, 207-234.

Tutar, Y. (2008). Syn, anti, and finally both conformations of cyclic AMP are involved in the CRP- dependent transcription initiation mechanism in E. coli lac operon. Cell Biochemistry and Function 26, 399-405.
How to Cite
Pliego-Arreaga, R., Regalado, C., Amaro-Reyes, A., & García-Almendárez, B. (2020). LACTOSE-INDUCED EXPRESSION OF RECOMBINANT TURNIP PEROXIDASE IN Escherichia coli. Revista Mexicana De Ingeniería Química, 12(3), 505-511. Retrieved from

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