info:eu-repo/semantics/article
Horseradish peroxidase production from Spodoptera frugiperda larvae: A simple and inexpensive method
Fecha
2010-06Registro en:
Targovnik, Alexandra Marisa; Romero, Lucía Virginia; Wolman, Federico Javier; Cascone, Osvaldo; Miranda, Maria Victoria; Horseradish peroxidase production from Spodoptera frugiperda larvae: A simple and inexpensive method; Elsevier; Process Biochemistry; 45; 6; 6-2010; 835-840
0032-9592
1359-5113
CONICET Digital
CONICET
Autor
Targovnik, Alexandra Marisa
Romero, Lucía Virginia
Wolman, Federico Javier
Cascone, Osvaldo
Miranda, Maria Victoria
Resumen
Horseradish peroxidase is used in many biotechnological fields including diagnostics, biocatalysts and biosensors. Horseradish peroxidase isozyme C (HRPC) was extracellularly expressed in Spodoptera frugiperda Sf9 cell culture and in intact larvae. At day 6 post-infection, the concentration of active HRPC in suspension cultures was 3.0 ± 0.1 μg per 1 × 106 cells or 3.0 ± 0.1 mg l-1 with a multiplicity of infection of 1 in the presence of 7.2 μM hemin. Similar yields were obtained in monolayer cultures. In larvae, the HRPC expression level was 137 ± 17 mg HRPC kg-1 larvae at day 6 post-infection with a single larvae thus producing approximately 41 μg HRPC. The whole larval extract was separated by ion exchange chromatography and HRPC was purified in a single step with a yield of 75% and a purification factor of 117. The molecular weight of recombinant HRPC was 44,016 Da, and its glycosylation pattern agreed with that expected for invertebrates. The Km and Vmax were 12.1 ± 1.7 mM and 2673 ± 113 U mg-1, respectively, similar to those of HRP purified from Armoracia rusticana roots. The method described in this study, based on overexpression of HRPC in S. frugiperda larvae, is a simple and inexpensive way to obtain high levels of active enzyme for research and other biotechnological applications. © 2010 Elsevier Ltd. All rights reserved.