FRACTIONATION OF FUNGAL PECTIC ENZYMES BY IMMOBILIZED METAL-ION AFFINITY-CHROMATOGRAPHY

A juice-clarifying fraction that will not produce methanol was obtaine d from a commercial pectic enzyme by separating the pectin lyase (EC4. 2.2.10) from pectinesterase (EC3.1.1.11). This was achieved by immobil ised metal ion affinity chromatography (IMAC) on Sepharose-iminodiacet ic acid-Cu(II). Pectin lyase did not bind to the chromatographic matri x at pH 8.0, while pectinesterase was retained and only eluted when th e pH of the buffer was brought down to 3.0. Polygalacturonase activity (EC3.2.1.15) was found in both fractions thus suggesting that IMAC ca n discriminate between two forms of that enzyme Both fractions have th e ability to clarify apple juice, the first without producing methanol . The behaviour of each of the above enzymes in IMAC suggests that pec tin lyase lacks accessible histidine residues, while pectinesterase co uld have one or more of them.