Title: 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 Food Hydrocolloids Authors
Submitted to: Food Hydrocolloids Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 14, 2014
Publication Date: February 19, 2014
Repository URL: http://dx.doi.org/10.1016/j.foodhyd.2014.01.022
Citation: Kim, Y., Williams, M.A., Tzen, J.T., Luzio, G.A., Galant, A.L., Cameron, R.G. 2014. 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 Food Hydrocolloids. Food Hydrocolloids Journal. 39:319-329. Interpretive Summary: Pectin is a natural polysaccharide found in plant cell walls. It is widely used in numerous food products, personal care and medical products. Pectin use is rapidly increasing largely due to the need for texturants in low fat food products and the increased popularity of acidified dairy beverages. The functional properties of pectins are highly dependent on their molecular structure, such as pattern and degree of demethylesterification (DM). Even though commercial pectins are conventionally classified by their DM into high (> 50% DM) or low methoxyl pectin (< 50% DM), recent studies have revealed that the pattern of methyl esterification has important commercial implications due to its impacts on the functional properties of pectins. Blockwise demethylesterification observed with plant enzymes is more likely to produce pectins that cross link significantly with calcium than random demethylesterification from chemical saponification and fungal enzymes. We used novel acidic form of a recombinant plant enzyme, whose gene has been inserted into a fungal expression vector, to demethylesterify a model pectin and then characterized the nanostructural changes we introduced. We also tested the gel forming properties of these pectins. We found we could manipulate the average size of a demethylesterified block and the average number of such blocks per molecule by choosing the target DM and the enzymatic reaction conditions. We also could correlate the resulting functional properties with the nanostructural changes we created.
Technical Abstract: A novel, 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 (BS) and number of such blocks per molecule (BN) differed depending on the degree of methylesterification (DM) and reaction pH (P < 0.05). Significant increases in BS and BN 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 BS 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 BS = 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. Pearson’s correlation displayed significant correlation coefficients between BS, BN, DBabs, DM, and G'. The results suggest the possibility to control BS and to produce a uniform population of demethylesterified pectin molecules, particularly in acidic environments where most basic plant PMEs are less inactive.