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

摘要

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 (PME) having different modes of action. A fungal PME from Aspergillus aculeatus (Aa-PME), with a pseudo-random mode of action, was used to deme-thylesterify a extremely high methylesterified HG (DM 94%, average degree of polymerization 246) by reducing the degree of methylesterification (DM) from 94% to either 70% or 80%. A second demethy-lesterification step, to 50% DM, was performed using a processive PME from Carica papaya (CpL-PME). Introduced demethylesterified blocks were released either by exhaustive or limited endo polygalacturonase (EPG) digestion. Degree of blockiness (DB), absolute degree of blockiness (DBabs), average demethylesterified block size ((BS) over bar) and number of average sized demethylesterified blocks per molecule ((BN) over bar) were estimated. (BS) over bar and (BN) over bar as well as DB/DBabs differed depending on the initial DM reduction by AaPME, the number of activity units of CpLPME used and the reaction pH (P 0.05). Consecutive demethylesterification of HG by AaPME to 80% DM and then by CpLPME to 50% DM at pH 4.5 produced significantly longer oligomer blocks compared to Aa-PME demethylesterification to 70% DM followed by CpL-PME to 50% DM at pH 7.0. Limited EPG digestion released nearly intact demethylesterified blocks and the released oligomers were coupled with in silico modeling. Resulting oligomer distribution corresponded to the in silico mode of action representing contiguous demethylesterification of 10 GalA residues rather than that of random or complete block-wise demethylesterification. Calcium-mediated gels of the modified HGs displayed G' higher than G '' values and both moduli differed significantly according to the amount of CpLPME applied even though their final DMs were identical. These results suggest the possibility of controlling (BS)over bar and engineering a population of demethylesterified pectin molecules with specified demethylesterified (BS) over bar and functional properties. (C) 2017 Elsevier Ltd. All rights reserved.