Study of variables involved in fungal pectic enzyme fractionation by immobilized metal ion affinity chromatography

The effects of the pH, composition and ionic strength of the chromatographic buffer and immobilized metal on the fractionation of a commercial pectic enzyme were studied using immobilized metal ion affinity chromatography. The best performance of the system was achieved with Cu2+ as the immobilized metal, 20 mM sodium phosphate (pH 7.0 containing 100 mM NaCl) as the adsorption buffer and 100 mM sodium acetate (pH 3.0 containing 100 mM NaCl) as the elution buffer. Under these conditions, pectinesterase was fully retained by the chromatographic matrix while the fraction passing through - containing pectin lyase - allows clarification of fruit juice without methanol production. The retained pectinesterase and the polygalacturonase contained in the commercial pectic enzyme preparation can be eluted quantitatively and applied to other industrial uses. From equilibrium adsorption isotherms, the maximum capacity of Chelating Sepharose for pectinesterase under the above conditions was 2990 +/- 190 U/ml at 20 degrees C and 2940 +/- 170 U/ml at 30 degrees C, while K-d values were 27.1 +/- 5.3 U/ml and 57.6 +/- 9.5 U/ml, respectively. From breakthrough curves, the optimum linear flow rate was 0.80 cm/min with a commercial preparation concentration at 100 mg/ml.