WOLMAN Federico Javier
congresos y reuniones científicas
Purification and characterization of horseradish peroxidase from Spodoptera Frugiperda larvae
A. M. TARGOVNIK; L. V. ROMERO; F. J. WOLMAN; O. CASCONE; M. V. MIRANDA
San Miguel de Tucuman, Tucuman, Argentina
Congreso; SAIB 2009, 45 Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2009
Horseradish peroxidase is useful in many biotechnological fields, including diagnostics, biocatalysis and biosensors. The aim of this work is to study different purification strategies for the recovery of recombinant horseradish peroxidase (HRPr) directly from Spodoptera frugiperda larva extract. The cDNA supports two tags (6XHis and 6XArg) and was cloned under the polyhedrin promoter and the signal peptide sequence GP67. Spodoptera frugiperda larvae were injected with 50 µl of recombinant baculovirus stock (1.4x107 pfu.ml-1). HRPr expression level was 137 ± 17 mg HRPC Kg-1 larvae at day 6 post infection. HRPr produced by a single larva was around 41 μg. The whole larvae extract was directly subjected to ion exchange chromatography on SP-Sepharose and HRPr was purified in only one step with a yield of 75% and a purification factor of 117. On other hand, HRPr was also purified by inmobilised metal ion affinity chromatography on Ni-Sepharose with a yield of 86% and a purification factor of 29. Both purification methods yielded virus-free enzyme. HRPr glycosylation pattern agrees with that expected for invertebrates. The kinetic parameters using guaiacol as substrate at 20ºC were determined. The apparent Km was 12.1 ± 1.7 mM and Vmax was 2673 ± 107 U mg-1, similar to standard HRP. The process herein described is an excellent way to obtain high levels of active HRPr.