Resumo

MIRANDA, M.V. et al. Optimisation of peroxidase adsorption on concanavalin A-agarose. Lat. Am. appl. res. [online]. 2003, vol.33, n.2, pp.67-71. ISSN 0327-0793.

Many publications describe methods for peroxidase purification from plant material. When the goal was to obtain a high purity enzyme every purification method included an affinity chromatography step using Concanavalin A as the ligand. However, the adsorption step was carried out under quite different conditions with regard to pH, ionic strength and metal cation content in the binding buffer, thus leading to a rather confuse situation. To establish the best conditions for purification of peroxidases from horseradish root (HRP) and soybean hull (SBP), equilibrium adsorption isotherms were fitted to the Langmuir-type model, where ionic strength, pH and cation concentration were chosen as the variables. For SBP, our results showed that Kd rounded 10^-6 M in all cases (pH 5.0 - 7.0, 1 and 5 mM Mn²⁺ / Ca²⁺, 0 - 0.75 M NaCl). For HRP, Kd ranged between 10^-5 M and 10^-6 M depending on the parameters. Under optimised binding conditions, 84.3% SBP was recovered after elution carried out with 0.74 M a -methyl-D-mannopyranoside and 0.37 M NaCl. For HRP, the recovery was lower (75%) and 0.36M a -methyl-D-mannopyranoside was necessary for the elution step.

Palavras-chave : Peroxidase; Adsorption; Concanavalin A-Agarose; Chromatography.

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