The molecular structure and multifunctionality of the cryptic plant polymer suberin

Abstract

Suberin, a plant polyester, consists of polyfunctional long-chain fatty acids and glycerol and is an intriguing candidate as a novel antimicrobial material. We purified suberin from cork using ionic-liquid catalysis during which the glycerol bonds that ensure the polymeric nature of suberin remained intact or were only partially cleaved-yielding the closest to a native configuration reported to date. The chemistry of suberin, both in situ (in cryogenically ground cork) and ex situ (ionic-liquid extracted), was elucidated using high-resolution one- and two-dimensional solution-state NMR analyses. Centrifugation was used to isolate suberin particles of distinct densities and their monomeric composition, assembly, and bactericidal effect, inter alia, were assessed. Analysis of the molecular structure of suberin revealed the relative abundance of linear aliphatic vs. acylglycerol esters, comprising all acylglycerol configurations and the amounts of total carbonyls (C[bond, double bond]O), free acid end groups (COOH), OH aliphatics, and OH aromatics. Suberin centrifuged fractions revealed generic physiochemical properties and monomeric composition ​and self-assemble into polygonal structures that display distinct degrees of compactness when lyophilized. Suberin particles-suberinsomes-display bactericidal activity against major human pathogenic bacteria. Fingerprinting the multifunctionality of complex (plant) polyesters such as suberin allows for the identification of novel polymer assemblies with significant value-added properties.

Keywords: Antimicrobial biopolymers; Cryogenic milling of plant polymers; Plant polyesters; Polymer self-assembly; Solution-state nuclear magnetic resonance; Suberin; Suberin particles.

Graphical abstract

Fig. 1

Microscopy imaging of the purified suberin. (a) Suberin in water formed aggregates of various sizes on the μm scale as observed by TEM imaging. (b) Corresponding freeze-dried samples formed ordered polygonal structures as observed by SEM imaging.

Fig. 2

Wide-ranging NMR spectral characterization of purified suberin. (a) The NMR ¹H and (b) ¹³C spectra, with inserts focusing on the aliphatic and aromatic regions; and the HSQC spectrum: (c) full and regions corresponding to (d) aliphatics, (e) glycerol CH-Acyl and CH/CH₂-X aliphatics, and (f) aromatics of the purified suberin. (g) The chemical structure of linear aliphatic esters and of acylglycerol esters comprising all possible configurations are presented. Text inserts in the HSQC spectra indicate the relative abundance (%) of the different acylglycerol configurations present within each sample as well as the ratio of aliphatic esters and acylglycerol esters. AcylGEs and LAEs stand for acylglycerol esters and linear aliphatic esters, respectively; TAG, DAG, and MAG stand for triacylglycerol, diacylglycerol, and monoacylglycerol, respectively. Some correlations (unlabeled) are uncertain or unidentified.

Fig. 3

Morphological and NMR chemical characterization of cryogenic milled cork and its residue after suberin removal. (a) Cryogenic milling was used to pulverize cork and its residue following suberin removal, (b–d) at levels that allow solubilization in heated DMSO for wide-ranging solution-state NMR spectral analyses. (a) SEM micrographs of cork and cork residues before and after cryomilling for 4 ​h show that the cork cell wall structure completely disappeared. (b) The ¹H NMR spectra of cork and cork residue show great chemical similarity to that of suberin. Comparison of the glycerol CH-Acyl region of the HSQC-NMR spectra of cork (c, gray) and of cork residues (d, gray) with that of suberin (c–d, red). Text inserts in the HSQC spectra indicate the relative abundance (%) of the different acylglycerol configurations present within each sample as well as the ratio of aliphatic esters and acylglycerol esters. AcylGEs and LAEs stand for acylglycerol esters and linear aliphatic esters, respectively; TAG, DAG, and MAG stand for triacylglycerol, diacylglycerol, and monoacylglycerol, respectively.

Fig. 4

Morphological and wide-ranging NMR spectral characterization of suberin centrifuged fractions. Suberin centrifuged fractions were obtained by sequential collection of pellets formed at defined centrifugation forces, namely 96, 385, 865, and 1538 ​g (plus a 2452 ​g minor fraction that accounts for less than 1% wt). (a) Their morphology was analyzed by SEM, (b–c) and their chemistry by wide-ranging NMR. (a) SEM imaging of the freeze-dried samples showed that they all formed ordered polygonal-like structures. (b) Their ¹H NMR spectra signatures were virtually identical, (c) but the glycerol CH-Acyl region of their corresponding HSQC-NMR spectra revealed extant differences in the relative abundance (%) of the distinct acylglycerol configurations. Text inserts in the HSQC spectra indicate the relative abundance (%) of the different acylglycerol configurations present within each sample as well as the ratio of aliphatic esters and acylglycerol esters. AcylGEs and LAEs stand for acylglycerol esters and linear aliphatic esters, respectively; TAG, DAG, and MAG stand for triacylglycerol, diacylglycerol, and monoacylglycerol, respectively.

Fig. 5

Suberin activity against E. coli and S. aureus. Following exposure of (a) E. coli and (b) S. aureus cells to suberin at concentrations of 0.25–2 ​μg ​mL⁻¹ (11 ​h), their morphology and viability were visualized by phase-contrast (arrows highlight the filamentous phenotype of E. coli cells, a) and fluorescence microscopy using Texas Red filter (dead cells show red fluorescence due to propidium iodide labeling, a–b), respectively. Controls are also shown (without suberin and a mixture of hydrolysate cork monomers). The scale bar in all images is 5 ​μm.