Demethylation of a model homogalacturonan with a salt-independent pectin methylesterase from citrus: I. Effect of pH on demethylated block size, block number and enzyme mode of action

A model homogalacturonan (HG) was used to produce a demethylated series by reacting it with a citrus salt-independent pectin methylesterase (PME) at pH 4.5 and 7.5. HGs with a predicted degree of methylesterification (DM) of 90%, 80%, 70%, 60% and 50% were produced. HG structural properties and PME mode of action were probed by performing a limited digest with an endo-polygalacturonase. Liberated demethylated blocks (DMB) were separated and quantified by HPAEC coupled to an evaporative light scattering detector. A significant increase in maximum and average size of released DMBs was observed between 80% and 70% DM at pH 7.5 and between 70% and 60% at pH 4.5. The observed distribution of fragment lengths was compared to theoretical modeled distributions. A multiple-attack mechanism with a degree of processivity (p) of p = 1 at pH 4.5 and p = 10 at pH 7.5 modeled the observed shorter fragments well, while in all cases the amount of larger fragments detected was more successfully described by a single-chain mechanism.