Heterologous expression and biochemical characterization of a highly active and stable chloroplastic CuZn-superoxide dismutase from Pisum sativumReportar como inadecuado

Heterologous expression and biochemical characterization of a highly active and stable chloroplastic CuZn-superoxide dismutase from Pisum sativum - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Biotechnology

, 15:3

First Online: 08 February 2015Received: 26 November 2014Accepted: 27 January 2015DOI: 10.1186-s12896-015-0117-0

Cite this article as: Tuteja, N., Mishra, P., Yadav, S. et al. BMC Biotechnol 2015 15: 3. doi:10.1186-s12896-015-0117-0


BackgroundCuZn-Superoxide dismutase SOD is a unique enzyme, which can catalyzes the dismutation of inevitable metabolic product i.e.; superoxide anion into molecular oxygen and hydrogen peroxide. The enzyme has gained wide interest in pharmaceutical industries due to its highly acclaimed antioxidative properties. The recombinant expression of this protein in its enzymatically active and stable form is highly desired and hence optimization of culture conditions and characterization of the related biochemical properties are essential to explore the significance of the enzyme in physiological, therapeutic, structural and transgenic research.

ResultsHigh-level expression of the chloroplastic isoform of Pisum sativum CuZn-SOD was achieved at 18°C, upon isopropyl β-D-1-thiogalactopyranoside induction and the process was optimized for maximum recovery of the protein in its soluble enzymatically active form. Both crude and purified protein fractions display significant increase in activity following supplementation of defined concentration Cu CuSO4 and Zn ZnSO4. Yield of the purified recombinant protein was ~ 4 mg L of culture volume and the bacterial biomass was ~ 4.5 g L. The recombinant pea chloroplastic SOD was found to possess nearly 6 fold higher superoxide dismutase activity and the peroxidase activity was also 5 fold higher as compared to commercially available CuZn-superoxide dismutase. The computational, spectroscopic and biochemical characterization reveals that the protein harbors all the characteristics features of this class of enzyme. The enzyme was found to be exceptionally stable as evident from pH and temperature incubation studies and maintenance of SOD activity upon prolonged storage.

ConclusionsOverexpression and purification strategy presented here describes an efficient protocol for the production of a highly active and stable CuZn-superoxide dismutase in its recombinant form in E. coli system. The strategy can be utilized for the large-scale preparation of active CuZn-superoxide dismutase and thus it has wide application in pharmaceutical industries and also for elucidating the potential of this protein endowed with exceptional stability and activity.

KeywordsHeterologous expression CD spectroscopy Homology modelling Ni-NTA purification Peroxidase activity Superoxide dismutase AbbreviationsCDCircular Dichroism



DTPADiethylenetriamine pentaacetic acid

E.coliEscherichia coli

H2O2Hydrogen peroxide

IPTGIsopropyl β-D-1-thiogalactopyranoside

PschSODPisum sativum SOD II

KCNPotassium cyanide

NaN3Sodium azide

SODSuperoxide dismutase

Panchanand Mishra and Sandep Yadav contributed equally to this work.

Electronic supplementary materialThe online version of this article doi:10.1186-s12896-015-0117-0 contains supplementary material, which is available to authorized users.

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Autor: Narendra Tuteja - Panchanand Mishra - Sandep Yadav - Marjan Tajrishi - Sudhir Baral - Surendra Chandra Sabat

Fuente: https://link.springer.com/

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