Characterization of non-esterified galacturonic acid sequences in pectin with endopolygalacturonase

A method was developed that enabled the study of non-esterified galacturonic acid sequences (so-called blocks) in pectin. Endopolygalacturonase of Kluyveromyces fragilis was used to extensively degrade pectin, and the composition of the galacturonic acid molecules produced was determined with high-performance anion-exchange chromatography at pH 5. With this technique, the amount of non-esterified mono-, di-, and trigalacturonic acid released was determined. In addition, the relative amounts of methyl-esterified oligomers--up to 10 galacturonic acid residues could be observed. By comparing the percentages of non-esterified mono-, di-, and trigalacturonic acids released, pectins with large enzyme-degradable blocks could be distinguished from pectins with small enzyme-degradable blocks. High percentages of mono- and digalacturonic acid were found for pectins containing small non-esterified blocks. The total area of all peaks corresponding to methyl-esterified oligomers was found to be indicative for the distribution of these blocks. The higher the ratio of the methyl- to non-esterified peak areas, the more closely associated blocks are present. Randomly esterified pectins, with degrees of methyl esterification of 50 and higher, contained smaller, more clustered blocks than commercial extracted pectins of comparable degrees of esterification. The approach developed enables a very detailed study of the methyl-ester distribution of pectin to be carried out and is a very important addition in the study of the functional behavior of this complex polymer.