Structural and functional effects of manipulating the degree of methylesterification in a model homogalacturonan with a pseudo-random fungal pectin methylesterase followed by a processive methylesterase

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

Submitted to: Food Hydrocolloids
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/20/2017
Publication Date: 2/3/2018
Citation: Kim, Y., Cameron, R.G., Williams, M.A., Luzio, G.A. 2018. Structural and functional effects of manipulating the degree of methylesterification in a model homogalacturonan with a pseudo-random fungal pectin methylesterase followed by a processive methylesterase. Food Hydrocolloids. 77:879-886.

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. Fungal pectin methylesterase introduce unmethylesterified galacturonic acid residues in a random fashion while plant pectin methylesterases introduce them in a block fashion. The objectives of this study were to characterize the nanostructural features introduced during demethylesterification of a model homogalacturonan with a fungal pectin methylesterase followed by a plant pectin methylesterase. The average demethylesterified block size and number of such blocks per molecule differed depending on the initial degree of methylesterification produced by the fungal pectin methylesterase, the amount of the plant pectin methylesterase used and the reaction pH. A processive, multiple attack mode of action for this combination of pectin methylesterases best explained the distribution of unmethylesterified galacturonic acid blocks introduced into the pectin molecules. Functional properties differed depending on several factors. The results suggest the possibility to control average demethylesterified block size and to engineer a population of demethylesterified pectin molecules with specified block sizes and functional properties.

Technical Abstract: We explored the possibility of controlling charge distribution in the homogalacturonan regions of pectin to produce a population of demethylesterified molecules with desirable functional properties by utilizing consecutive treatments with pectin methylesterases having different modes of action. A fungal pectin methylesterase from Aspergillus aculeatus, with a pseudo-random mode of action, was used to demethylesterify a model homogalacturonan by reducing the degree of methylesterification from 94% to either 70% or 80%. A second demethylesterification step, to 50% degree of methylesterification, was performed using a progressive pectin methylesterase from Carica papaya. Introduced demethylesterified blocks were released either by exhaustive or limited endo polygalacturonase digestion. Degree of blockiness, absolute degree of blockiness, average demethylesterified block size and number of average sized demethylesterified blocks per molecule were estimated. Block size and block number as well as degree of blockiness and absolute degree of blockiness differed depending on the initial degree of methylesterification reduction by the fungal pectin methylesterase, the number of activity units of papaya pectin methylesterase used and the reaction pH (P<0.05). Consecutive demethylesterification of homogalacturonan by fungal pectin methylesterase to 80% DM and then by papaya pectin methylesterase to 50% degree of methylesterification at pH 4.5 showed significantly longer oligomer blocks compared to fungal pectin methylesterase demethylesterification to 70% DM followed by papaya pectin methylesterase to 50% degree of methylesterification at pH 7.0. Limited endo polygalacturonase digestion, releasing nearly intact demethylesterified blocks, coupled with in silico modeling describes a distribution of charge that would be obtained using a single progressive pectin methylesterase having a multiple attack mode of action. Calcium-mediated gels of the modified homogalacturonans displayed a solid modulus higher than liquid modulus values and both moduli differed significantly according to the amount of papaya pectin methylesterase applied even though their final degree of methylesterifications were identical. These results suggest the possibility of controlling block size and engineering a population of demethylesterified pectin molecules with specified demethylesterified and functional properties.