Chromatographic separation of orange pectinesterase isoenzymes on pectates with different degrees of cross-linking

Abstract

A selected sodium pectate preparation, suspended in an alkaline mixture of ethanol and water, was cross-linked with epichlorohydrin. By varying the amount of epichlorohydrin in the reaction mixture, preparations were obtained with different degrees of cross-linking. The preparations were used as chromatographic media for the separation and purification of two orange pectinesterase isoenzymes. The binding of the isoenzymes was found to depend on the degrees of cross-linking of the pectate. On pectate with an intermediate degree of cross-linking (0.46) both isoenzymes were retained and subsequently eluted, with a sodium chloride gradient, as separated peaks with a 7- to 8-fold increase in specific activity. Both pectinesterase isoenzymes saponified 93% esterified pectin with a high degree of cross-linking. It is thought that the binding of these enzymes to cross-linked pectate involves biospecific affinity.