Lactose-induced expression of recombinant turnip peroxidase in Escherichia coli
Expresión inducida por lactosa de peroxidasa de nabo recombinante en Escherichia coli
R. Pliego-Arreaga, C. Regalado, A. Amaro-Reyes y B.E. García-Almendárez*
DIPA, PROPAC, Facultad de Química. Universidad Autónoma de Querétaro. C.U., Cerro de las Campanas s/n (Col. Las Campanas 76010, Querétaro, Qro., México. * Autora para la correspondencia. E-mail: blancag31@gmail.com.
]]> Received August 8, 2013
Abstract
Turnip (Brassica napus L. var. purple top white globe) peroxidases (E.C. 1.11.1.7) 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.
Keywords: Brassica napus, peroxidase, expression, lactose, fermenter.
Resumen
Las peroxidasas (E.C. 1.11.1.7) de nabo (Brassica napus L. var. purple top White globe) pueden aplicarse libres o inmovilizadas para la degradación de compuestos fenólicos en aguas residuales además de su uso en kits de diagnóstico. El objetivo de este trabajo fue lograr una expresión heteróloga por inducción con lactosa de una isoforma de peroxidasa (PodC) usando Escherichia coli Rosetta 2 para producir peroxidasa de nabo recombinante a nivel fermentador. PodC recombinante contenida en cuerpos de inclusión se replegó bajo condiciones oxidativas y se purificó por cromatografía para obtener 36 mg L-1 de cultivo. La enzima tuvo una actividad específica de 1004 unidads ABTS mg-1 bajo condiciones optimas (pH 6, 45 °C). El Km para ABTS fue 0.38 mM y la eficiencia catalítica (kcat/Km) fue 5.4×106 s-1 M-1. El uso de lactosa como inductor y el escalamiento del proceso de fermentación puede reducir el costo de producción de PodC recombinante, que a su vez puede usarse en aplicaciones biotecnológicas.
Palabras clave: Brassica napus, peroxidasa, expresión, lactosa, fermentador.
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