Charged functional domains introduced into a modified pectic homogalacturonan by a cocktail of pectin methylesterases isozymes from sweet orange (Citrus sinensis L. Osbeck var. Pineapple)

Authors: KIM, YANG - Seoul National University; Cameron, Randall - Randy; WILLIAMS, MARTIN - Massey University; LEE, CHANG JOO - Macdiarmid Institute For Advanced Materials And Nanotechnology

Submitted to: Food Hydrocolloids
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/1/2019
Publication Date: 5/29/2019
Citation: Kim, Y., Cameron, R.G., Williams, M.A., Lee, C. 2019. Charged functional domains introduced into a modified pectic homogalacturonan by a cocktail of pectin methylesterases isozymes from sweet orange (Citrus sinensis L. Osbeck var. Pineapple). Food Hydrocolloids. 96:589-595. https://doi.org/10.1016/j.foodhyd.2019.05.049.
DOI: https://doi.org/10.1016/j.foodhyd.2019.05.049

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. The objectives of this study were to characterize the nanostructural features introduced during demethylesterification of a model homogalacturonan with a mixture of pectin methylesterase found in citrus fruits and to thereby elucidate their combined 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. It was found that a measure of the average size of demethylesterified blocks was a better predictor for the introduced nanostructure than an estimate of the absolute degree of blockiness. A processive, multiple attack mode of action for this mixture pectin methylesterases 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: Multiple pectin methylesterase isozymes present in Citrus sinensis L. Osbeck were applied as an enzyme cocktail to demethylesterify a model homogalacturonan with a degree of methylesterification of 91% to 50% or 70% 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 per molecule were observed in homogalacturonans of 50% compared to 70 % degree of methylesterification. Homogalacturonans demethylesterified to 50% degree of methylesterification at pH 7.0 showed significantly larger average demethylesterified block size compared to the one at pH 4.5 (P < 0.01). Degree of blockiness and absolute degree of blockiness, obtained using exhaustive endo polygalacturonase digestions, were highest in 50% degree of methylesterification pH 4.5 and lowest in 70% degree of methylesterification at pH 7.0, displaying a similar trend with the average demethylesterified block size, accordingly, a high significant correlation between the block parameters were was observed (P<0.05). However, no difference was observed in the Absolute Degree of Blockiness values between treatment pHs sharing similar degrees of methylesterification, meaning these could not efficiently separate the difference of demethylesterified blocks existing within the homogalacturonans which while average demethylesterified block size and number of such blocks per molecule could. 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, multiple attack mode of action best explains the distributions of blocks. The results suggesting the possibility to introduce designated size of blocks to demethylesterified pectin molecules by applying multiple pectin methylesterase enzymes within citrus.