Study of variables involved in horseradish and soybean peroxidase purification by affinity chromatography on concanavalin A agarose

M.V. MIRANDA; M.L. MAGRI; A.A. NAVARRO DEL CAÑIZO; O. CASCONE

PROCESS BIOCHEMISTRY

Elsevier

Año: 2002 vol. 38 p. 537 - 543

0032-9592

  Variables involved in adsorption and elution of horseradish and soybean seed peroxidases from a concanavalin A-Agarose matrix were studied. The effect of pH, ion strength and Ca2+/Mg2+ concentration on maximum capacity and dissociation constant was assessed through adsorption isotherms. The effect of flow rate and peroxidase concentration on dynamic capacity was assessed through breakthrough curves. For the elution step, NaCl and a-D-methylmannopyranoside concentrations were optimised. The best conditions for soybean peroxidase adsorption were pH 5.0 and 1 mM Ca2+/Mg2+ in the absence of salt, at a flow rate up to 3.2 cm/min and 3 mg/ml peroxidase concentration. Elution required the addition of 0.48 M a-D-methylmannopyranoside and 0.97 M NaCl. Under these conditions, dynamic capacity was 16.9 mg/ml matrix and purification yield, 84.3 %. In the case of horseradish peroxidase, the best adsorption conditions were pH 7.0, 5 mM ions and 0.75 M NaCl, at a flow rate up to 1.5 cm/min and a 4 mg/ml peroxidase concentration. Dynamic capacity was 9.6 mg/ml matrix, and elution required 0.36 M a-D-methylmannopyranoside to yield 75 % of enzyme. The dynamic-to-static capacity ratios were 0.71 and 0.62 for horseradish and soybean peroxidases, respectively.