ARS | Publication request: Demethylation of a model homogalacturonan with a citrus salt-independent pectin methylesterase: effect of pH on block size and number, enzyme mode of action and resulting functionality

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: December 26, 2006
Publication Date: N/A

Technical Abstract: A demethylation series with predicted degrees of methylesterification (DM) of 90, 80, 70, 60 and 50 % was produced from a model homogalacturonan (HG) by reacting it with a citrus salt-independent pectin methylesterase (PME) at pH 4.5 and 7.5. PME mode of action and HG structural properties were probed by performing a limited digest with an endo-polygalacturonase (EPG) to excise demethylated blocks (DEMB) of galacturonic acid (GA). HPAEC coupled to an evaporative light scattering detector was used to separate and quantify the EPG liberated DEMBs. Functionality of the demethylated HGs was determined using the Calcium Sensitive Pectin Ration (CSPR) and the rheological properties of Yield Point (YP) and Storage Modulus (G'). A significant increase in maximum (GAn) and average block size (B) of released DEMBs was observed between 80 % (GA14, B=5.3) and 70 % DM (GA42, B=10.2) at pH 7.5 and between 70 % (GA19, B=5.3) and 60 % (GA43, B=7.9) at pH 4.5. DEMBs > GA50 were observed in the pH 4.5 series at 50 % DM. While was greater in the pH 7.5 series, the average number of blocks per molecule was greater in the pH 4.5 series. 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 the amount of larger fragments detected in all cases was more successfully described by a single chain mechanism. Functionality, i.e. CSPR and rheological properties, was shown to be dependent on demethylation pH as well as DM. At pH 7.5–70 % DM the values for CSPR, YP and G' were 0.8 %, 0.042 Pa and 0.017 Pa respectively. At pH 4.5 – 70 % DM these values were 11.4 %, 0.171 Pa and 1.06 Pa respectively. These combined results suggest this PME may possess a variably processive mode of action, becoming more processive as the DM decreases, and that both size and number of DEMBs per molecule need to be considered for relating fine structure to functionality.