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

Authors: KIM, YANG - Seoul National University; WILLIAMS, MARTIN - Massey University; LUZIO, GARY - Former ARS Employee; Cameron, Randall - Randy

Submitted to: Food Hydrocolloids
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/3/2017
Publication Date: 3/6/2017
Citation: Kim, Y., Williams, M., Luzio, G., Cameron, R.G. 2017. 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. Food Hydrocolloids. 69:422-431. doi: 10.1016/j.foodhyd.2017.03.009.

Interpretive Summary: The functionality of pectin is known to depend mostly upon the amount and distribution of unmethylesterified galacturonic acid units in the linear, polymeric backbone of pectin molecules. Recent investigations have revealed that the distribution and size of blocks of contiguous unmethylesterified galacturonic acid units have more importance than overall degree of methylesterification. The gelling functionality of pectin is mainly determined by the ability to form stable junction zones, hence it is critical to have the availability of unmethylesterified galacturonic acid blocks long enough for cross-linking via calcium ion bridges. Pectin methylesterases are enzymes utilized to introduce these blocks of successive unmethylesterified galacturonic acid residues into a homogalacturonan region which can serve as junction zones in many food, cosmetic, and pharmaceutical applications. The presence of multiple forms of pectin methylesterase have been described from citrus fruit tissue. Interestingly, only one of the four pectin methylesterase isozymes separated from sweet orange fruit peel did not destabilize orange juice cloud while the other three pectin methylesterases destabilized the juice cloud at varying rates. The objectives of this study were to characterize the nanostructural features introduced during demethylesterification of a model homogalacturonan with the pectin methylesterase that did not destabilize orange juice cloud and to thereby elucidate its mode of action at different pHs. The average demethylesterified block size and number of such blocks per molecule differed depending on the degree of methylesterification and reaction pH. A processive, multiple attack mode of action for this pectin methylesterase best explained the distribution of unmethylesterified galacturonic acid blocks introduced into the pectin molecules. The results suggest the possibility to control average demethylesterified block size and to produce a uniform population of demethylesterified pectin molecules.

Technical Abstract: One of the four pectin methylesterase isozymes isolated from Citrus sinensis variety Valencia fruit was used to demethylesterify a model homogalacturonan to 30%, 50% and 70% degree of methylesterification at pH 4.5 and 7.0, respectively. Introduced demethylesterified blocks were released by a limited endo polygalacturonase digestion, separated and quantified by high performance anion exchange chromatography. The average demethylesterified block size and number of such blocks per molecule differed depending on the degree of methylesterification and reaction pH (P < 0.05). Significant increases in average demethylesterified block size and number were observed in homogalacturonans of 30% degree of methylesterification compared to higher degrees of methylesterification. Homogalacturonans demethylesterified at pH 4.5 showed significantly larger average demethylesterified block size compared to pH 7.5 (P < 0.01). Absolute degree of blockiness, obtained using exhaustive endo polygalacturonase digestions, displayed a linear relationship with the degree of methylesterification regardless of reaction pH (P < 0.001). The distribution of demethylesterified blocks released by the limited endo polygalacturonase digest was predicted by mathematical modeling and compared with the experimental results. The in silico modeled enzyme mode of action suggested that a processive (degree of processivity = 10), multiple attack mode of action best explains the block distributions. Pearson’s correlation displayed significant correlation coefficients between average demethylesterified block size, block number, absolute degree of blockiness, and degree of methylesterification manifesting the effectiveness of the block information gained from both exhaustive and limited endo polygalacturonase digestion to estimate the introduced block distribution pattern by pectin methylesterase. The results suggest the possibility to control average demethylesterified block size and to produce a uniform population of demethylesterified pectin molecules.