Investigation of the non-esterified galacturonic acid distribution in pectin with endopolygalacturonase

A method was developed that enabled the quantification of non-methyl-esterified galacturonic acid (GalA) sequences in pectin using enzymes. Endopolygalacturonase of Kluyveromyces fragiles was used to degrade pectin and the mono-, di-, and oligogalacturonides liberated were analyzed with high-performance anion-exchange chromatography at pH 5. With this technique non-methyl-esterified GalA residues could be distinguished from partially methyl-esterified oligomers. Non-esterified mono-, di-, and tri-galacturonic acid were predominantly released. The total amount of non-esterified GalA liberated was expressed as the percentage of the total number of non-esterified GalA present in pectin. For this percentage the term ‘degree of blockiness’ is introduced. Pectins that were sequentially de-esterified with tomato pectin methylesterase released large amounts of non-esterified GalA. The more methyl esters removed with pectin methylesterase, the more non-methyl-esterified GalA were liberated by endopolygalacturonase. Random methyl-esterified pectins liberated the lowest amounts of non-esterified GalA, even when the degree of methyl esterification was relatively low. The method reveals clear differences between pectins having the same degree of methyl esterification and different functional behavior.