Introduction and characterization of charged functional domains into an esterified pectic homogalacturonan by a citrus pectin methylesterase and comparison of its modes of action to other pectin methylesterase isozymes

Abstract

One of the four pectin methylesterase types isolated from Citrus sinensis var. Valencia fruit was used to demethylesterify a model homogalacturonan (HG) to 30%, 50% and 70% degree of methylesterification (DM) at pH 4.5 and 7.0, respectively. Introduced demethylesterified blocks (DMBs) were released by a limited endo-polygalacturonase (EPG) digestion, separated and quantified by HPAEC. Average DMB size (BS¯) and number of such blocks per molecule (BN¯) differed depending on final DM and reaction pH (P < 0.05). BS¯ and BN¯ were significantly higher in 30% DM HG than 50 and 70 DMs. pH 4.5 series showed significantly larger BS¯ compared to pH 7.5 series (P < 0.01). Distribution of DMBs released by limited EPG digest was predicted by mathematical modeling and in silico modeled processive (degree of processivity = 10), multiple attack mode of action best explains the experimental block distributions. Absolute degree of blockiness (DBabs) obtained from exhaustive EPG digestions, displayed a linear relationship with DM regardless of reaction pH (P < 0.001). Significant correlation coefficients between BS¯, BN¯, DBabs, and DM manifested the effectiveness of the block information gained from both EPG digestion to estimate DMB distribution pattern (P < 0.05). However, comparison of block distribution information of three isozymes revealed that difference in block pattern could be manifested by parameters from limited EPG digest (BS¯, BN¯) but not by those from exhaustive digest (DB/DBabs). The results suggested the possibility to control BS¯ and to customize specific population of demethylesterified pectin molecules using PME isozymes from Valencia orange.