Mobility of pectin methylesterase in pectin/cellulose gels is enhanced by the presence of cellulose and by its catalytic capacity

Estelle Bonnin,Camille Alvarado,Catherine Garnier,Brigitte Bouchet,Marie-Françoise Devaux,Frédéric Jamme,Marie-Jeanne Crépeau

doi:10.1038/s41598-019-49108-x

Estelle Bonnin, Camille Alvarado + Show 5 more

Open Access

https://doi.org/10.1038/s41598-019-49108-x

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Abstract

The pectin methylesterase action is usually studied in a homogeneous aqueous medium in the presence of a large excess of soluble substrate and water. However in the cell wall, the water content is much lower, the substrate is cross-linked with itself or with other polymers, and the enzyme has to diffuse through the solid matrix before catalysing the linkage breakdown. As plant primary cell walls can be considered as cellulose-reinforced hydrogels, this study investigated the diffusion of a fungal pectin methylesterase in pectin/cellulose gels used as cell wall-mimicking matrix to understand the impact of this matrix and its (micro) structure on the enzyme’s diffusion within it. The enzyme mobility was followed by synchrotron microscopy thanks to its auto-fluorescence after deep-UV excitation. Time-lapse imaging and quantification of intensity signal by image analysis revealed that the diffusion of the enzyme was impacted by at least two criteria: (i) only the active enzyme was able to diffuse, showing that the mobility was related to the catalytic ability, and (ii) the diffusion was improved by the presence of cellulose in the gel.

Highlights

Plant cell walls consist mainly of polymer assemblies, whose structure and interactions vary depending on plant genetics and development
As we demonstrated previously that the pectin methylesterases (PME) from Aspergillus aculeatus diffused faster than the orange PME, the fungal PME was applied on pectin/cellulose gels prepared with increasing contents in cellulose
The binary gels were prepared in the presence of calcium, which resulted in a weak pectin/Ca2+ gel and in an interpenetrating network of cellulose and pectin

Summary

Introduction

Plant cell walls consist mainly of polymer assemblies (pectin, hemicelluloses, cellulose and proteins), whose structure and interactions vary depending on plant genetics and development. Plant PME act according to a single-chain and multiple attack mechanism, behaving as a processive enzyme and releasing blockwise distributed free GalA4,12–15 In all these studies, the modes of action were determined on a soluble substrate in dilute solutions, i.e. in a large excess of substrate and in homogenous medium. Both experiments were carried out on the low-energy beamline DISCO at the French synchrotron SOLEIL on a full-field fluorescence microscope, and gave the opportunity to follow unlabelled enzymes at high resolution and in real time From this background, we aim to investigate the PME diffusion in a cell wall-mimicking matrix and to understand the impact of this matrix and its (micro) structure on the efficiency and the mode of action of the enzyme.

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