Preparation of Cross-linked Pectate and Its Application to the Purification of Endopolygalacturonase of Kluyveromyces fragilis

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An improved method for preparing a cross-linked pectate (CLPA) and its application to the purification of endopolygalacturonase as an affinity adsorbent are shown. The addition of dimethyl sulfoxide was found to be very effective for the cross-linking reaction of pectate with epichlorohydrin. The yield of CLPAwas satisfactory in the presence of the organic solvent at 15 to 35%, maximal at about 25% but markedly decreased beyond this range. Endopolygalacturonase, one of the enzymes having an affinity for CLPA, was rapidly purified from Kluyveromycesfragilis culture fluids with a high yield using the CLPAcolumn. The purified enzyme exists in three different charged forms and in two molecular-sizes. The values of their isoelectric points were in fair agreement with those of the same enzymein the literature.
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