Title: 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 Authors
Submitted to: Meeting Proceedings
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 12, 2007
Publication Date: January 2, 2008
Citation: Cameron, R.G., Luzio, G.A., Goodner, K.L., Williams, M.A. 2008. 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. Gums and Stablisers for the Food Industry. 14:141-152. Interpretive Summary: The mode of action of the citrus pectin demethylating enzyme appears to have a variable degree of processivity, on average producing only relatively small demethylated blocks of pectin in a model system. The size of the demethylated blocks produced by the enzyme was dependent on the pH of the reaction buffer and the final degree of methylation. Similar block sizes could be introduced by manipulating both pH and endpoint degree of methylation so that an optimized process could be engineered to achieve the desired structural/functional property for predicted pectin polymers. Calcium sensitivity and rheological properties associated with functionality could be manipulated by the amount of demethylation and the pH at which it was conducted. This flexibility has the potential to eliminate potential problems that might be associated with undesirable pHs such as side reactions or corrosion. The ability to obtain the same end product under varying conditions may be very helpful for custom tailoring a pectin product for a commercial process.
Technical Abstract: A demethylation series with degrees of methylesterification (DM) of 90, 80, 70, 60 and 50 % was produced by reacting a model homogalacturonan (HG) 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 and average block size (B) of released DEMBs was observed between 80 % and 70 % DM at pH 7.5 and between 70 % and 60 % at pH 4.5. DEMBs > GA50 were observed in the pH 4.5 series at 50 % DM. While B 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. 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.