Fractionation of fungal pectic enzymes by immobilised metal ion affinity chromatography

A juice-clarifying fraction that will not produce methanol was obtained from a commercial pectic enzyme by separating the pectin lyase (EC4.2.2.10) from pectinesterase (EC3.1.1.11). This was achieved by immobilised metal ion affinity chromatography (IMAC) on Sepharose-iminodiacetic acid-Cu(II). Pectin lyase did not bind to the chromatographic matrix at pH 8.0, while pectinesterase was retained and only eluted when the 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 can discriminate between two forms of that enzyme. Both fractions have the ability to clarify apple juice, the first without producing methanol. The behaviour of each of the above enzymes in IMAC suggests that pectin lyase lacks accessible histidine residues, while pectinesterase could have one or more of them.