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. (C) 1999 Elsevier Science Ltd. All rights reserved.