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Title: 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

Author
item Cameron, Randall - Randy
item Goodner, Kevin
item Luzio, Gary
item WILLIAMS, MARTIN A. - MASSEY UNIV, NEW ZEALAND

Submitted to: Carbohydrate Polymers
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/12/2007
Publication Date: 1/2/2008
Citation: Cameron, R.G., Goodner, K.L., Luzio, G.A., Williams, M.K. 2008. 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. Carbohydrate Polymers. 71:287-299.

Interpretive Summary: The mode of action of the citrus salt-independent pectin methylesterase appears to have limited processivity, on average producing only relatively small demethylated blocks 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. This flexibility has the potential to eliminate 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 commercial process.

Technical Abstract: A model homogalacturonan (HG); composed of galacturonic acid (94-97 %) and galactose (3-6 %), having a 94 % degree of esterification (DE) was used to produce a demethylated HG series by reacting it with the salt-independent pectin methylesterase (PME) from citrus fruit at pH 4.5 and 7.5. HGs with a predicted degree of methylation of 90, 80, 70, and 60 % were produced. Structural properties of the demethylated HG and PME mode of action were probed by performing a limited digest with an endo-polygalacturonase from Aspergillus aculeatus. Liberated demethylated blocks (DB) of galacturonic acid were separated and quantified by high performance anion exchange chromatography coupled to an evaporative light scattering detector. With demethylation at pH 7.5 the longest DB size increased dramatically from 19 at 80% DE to 47 at 70% DE while the average DB size increased from 5 to 10 and the average number of DBs per molecule remained relatively constant increasing only from 1.7 to 2.0. A similar increase in size of the longest DB and average DB size was observed in the pH 4.5 demethylated HG, only at the transition from 70% to 60% DE. Deviation from a hypothetical model for a single chain mode of action and the dominate occurrence of oligomers with a DP < 9 suggest a limited degree of processivity for this PME.