ARS | Publication request: Nanostructural modification of a model homogalacturonan with a novel pectin methylesterase: Effects of pH on nanostructure, enzyme mode of action and substrate functionality

Submitted to: Food Hydrocolloids Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 20, 2013
Publication Date: April 1, 2013
Citation: Kim, Y., Williams, M.A., Galant, A.L., Luzio, G.A., Savary, B., Vasu, P., Cameron, R.G. 2013. Nanostructural modification of a model homogalacturonan with a novel pectin methylesterase: Effects of pH on nanostructure, enzyme mode of action and substrate functionality. Food Hydrocolloids Journal. 33:132-141.

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 the pectic homogalacturonans (HG’s) 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 an inexpensive, commercially available enzyme 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 pectin methylesterase (CpL-PME) present in a commercial papain preparation was used to modify the amount and distribution of charge in a model pectic homogalacturonan (HG) at pH 4.5 and pH 7.5. Introduced negatively charged demethylesterified blocks (DMB) were excised as oligomers with a limited endo polygalacturonase (EPG) digestion, and separated and quantified by high performance anion exchange chromatography (HPAEC). Exhaustive EPG digestion was also performed and the concentrations of mono-, di- and tri-galacturonic acid (GalA) produced were estimated. The CpL-PME mode of action was modeled while the DMB nanostructure and the resulting functionality of the modified pectin were characterized. The results indicated that average DMB size (BS) and number of these “average” blocks per molecule (BN) increased according to the degree of methylesterification (DM) decrease (P<0.05). BS differed depending on the reaction pH at each DM. Absolute degree of blockiness (DBabs) increased as DM decreased and demonstrated a linear relationship with the DM at both reaction pH values. Significant positive correlations between DBabs and BS were observed for both pH series (P<0.05). The enzyme mode of action, modeled in silico, demonstrates a processive multiple attack mechanism. Functionally, the storage modulus of the processed substrate increased as DM decreased, and as DBabs and BS increased with highly significant correlations (P<0.001). These results indicate the feasibility of engineering pectin nanostructure and functionality with CpL-PME.