Characterization of charged functional domains introduced into a modified pectic homogalacturonan by an acidic plant pectin methylesterase (Ficus awkeotsang Makino) and modeling of enzyme mode of action

An acidic plant pectin methylesterase from Ficus awkeotsang achenes (FaPME) was used to demethylesterify a model homogalacturonan (HG) at pH 4.5 and 7.5. Introduced demethylesterified blocks (DMBs) were released by a limited endo-polygalacturonase (EPG) digestion, separated and quantified by HPAEC. The average DMB size ( B S ¯ ) and number of such blocks per molecule ( B N ¯ ) differed depending on the degree of methylesterification (DM) and reaction pH (P < 0.05). Significant increases in B S ¯ and B N ¯ were observed in HGs of 30% DM compared to higher DMs. HGs demethylesterified to 30% and 50% DM at pH 4.5 showed significantly larger B S ¯ compared to pH 7.5. Absolute degree of blockiness (DBabs), obtained using exhaustive EPG digestions, displayed a linear relationship with the DM regardless of reaction pH (P < 0.001). The distribution of DMBs released by the limited EPG digest was predicted by mathematical modeling and compared with the experimental results. The in silico modeled enzyme mode of action suggested that a random, multiple chain, non-processive mode of action best explains the distributions of small blocks ( B S ¯ ≥ 11 ) and a processive multiple attack mechanism best explains the distributions of longer blocks. Decreasing the DM of the HGs by the FaPME increased the G′ and G″ of calcium-mediated gels. Pearsonʹs correlation displayed significant correlation coefficients between B S ¯ , B N ¯ , DBabs, DM, and G′. The results suggest the possibility to control B S ¯ and to produce a uniform population of demethylesterified pectin molecules, particularly in acidic environments where most basic plant PMEs are less active.