endo-Polygalacturonanase M2
(Aspergillus aculeatus)

In this study, tailored-made citrus pectin-derived compounds were produced through controlled enzymatic and/or chemical modifications of commercial citrus pectin with different degrees of methylesterification (DM) and similar average molecular weight (M_W). In the first treatment, degradation of the citrus pectin (CP) materials by endo-polygalacturonase (EPG) yielded pectins with average M_w’s (between 2 and 60 kDa). Separation and identification of the oligosaccharide fraction present in these samples, revealed the presence of non-methylesterified galacturonic acid oligomers with degree of polymerization (DP) 1-5. In the second treatment, exploiting the combined effect of EPG and pectin lyase, compounds with M_W between 2 and 21 kDa, containing methylesterified and non-methylesterified polygalacturonans (DP 1-6), were generated. Finally, CP was sequentially modified by chemical saponification and the action of EPG. A sample of DM 11% and M_W 2.7 kDa, containing POS (DP 1-5), was produced. Diverse pectin-derived compounds were successfully generated for further studies exploring their functionality.

Tau, a microtubule-associated protein expressed in mature neurons, interacts with tubulin to promote the assembly and stabilization of microtubules. However, abnormally hyperphosphorylated tau dissociates from microtubules and self-aggregates. Tau aggregates, including paired helical filaments and neurofibrillary tangles, promote neuronal dysfunction and death and are the defining neuropathological feature of tauopathies. Therefore, suppressing tau aggregation or stimulating the dissociation of tau aggregates has been proposed as an effective strategy for treating neurodegenerative diseases associated with tau pathology such as Alzheimer’s disease (AD) and frontotemporal dementia. Interestingly, ginsenosides extracted from Panax ginseng reduced the hippocampal and cortical expression of phosphorylated tau in a rat model of AD. However, no studies have been conducted into the effect of red ginseng (RG) and its components on tau pathology. Here, we evaluated the effect of Korean red ginseng extract (KRGE) and its components on the aggregation and disassociation of tau. Using the thioflavin T assay, we monitored the change in fluorescence produced by the aggregation or disassociation of tau K18, an aggregation-prone fragment of tau₄₄₁ containing the microtubule-binding domain. Our analysis revealed that KRGE not only inhibited tau aggregation but also promoted the dissociation of tau aggregates. In addition, the KRGE fractions, such as saponin, nonsaponin, and nonsaponin fraction with rich polysaccharide, also inhibited tau aggregation and promoted the dissociation of tau aggregates. Our observations suggest that RG could be a potential therapeutic agent for the treatment of neurodegenerative diseases associated with tauopathy.

Endopolygalacturonases characterized until now have either low working temperatures, working pH in acidic range, high Michaelis-Menten constant (K_m), or a high production cost. These characteristics are a hurdle in the industrial applications of these endopolygalacturonases. The purpose of this work was to characterize a novel endopolygalacturonase produced by Bacillus licheniformis IEB-8. Phylogenetic analysis of Bacillus licheniformis IEB-8 showed that the isolate was unique. Citrus peels were used as the only nutrient source for the growth of Bacillus licheniformis IEB-8, allowing a cheap production of endopolygalacturonase. All the synthetic carbon sources showed a negative impact on the production of endopolygalacturonase, while ammonium sulfate enhanced its production. Among different metal ions, Zn⁺² showed a negative effect while Mg⁺² and Ca⁺² did not have any significant effect on the endopolygalacturonase activity. A Lineweaver-Burk plot was prepared for the characterization of the kinetic parameters including K_m and V_max, which were 0.45 mg/mL and 285.7 µM/min, respectively. A comprehensive comparison of the endopolygalacturonase from this study with the available literature indicated that it is better than the reported and commercially available endopolygalacturonases in having the optimum working temperature of 55°C, a low Km of 0.57 mg/mL, and pH of 7 to 8, which indicated its novelty.

Background and aims: Tolerance to soil acidity was studied in two species of Ericaceae that grow in mine-contaminated soils (S Portugal, SW Spain) to find out if there are interspecific variations in H⁺ tolerance which might be related to their particular location. Methods: Tolerance to H⁺ toxicity was tested in nutrient solutions using seeds collected in SW Spain. Plant growth and nutrient contents in leaves, stems and roots were determined. Viability tests and proton exchange were studied in roots exposed, short-term, to acidic conditions. Membrane ATPase activity and the cell-wall pectic polysaccharide domain rhamnogalacturonan-II (RG-II) were analysed to find out interspecific differences. Results: Variation in survival, growth and mineral composition was found between species. The H⁺-tolerant species (Erica andevalensis) showed greater concentration of nutrients than E. australis. Very low pH (pH 2) produced a significant loss of root nutrients (K, P, Mg) in the sensitive species. Root ATPase activity was slightly higher in the tolerant species with a correspondingly greater H⁺ efflux capacity. In both species, the great majority of the RG-II domains were in their boron-bridged dimeric form. However, shifting to a medium of pH 2 caused some of the boron bridges to break in the sensitive species. Conclusions: Variation in elements linked to the cell wall-membrane complex and the stability of their components (RG-II, H⁺-ATPases) are crucial for acid stress tolerance. Thus, by maintaining root cell structure, active proton efflux avoided toxic H⁺ build-up in the cytoplasm and supported greater nutrient acquisition in H+-tolerant species.

Pectin oligosaccharides (POS) have been indicated as a new class of potential prebiotic compounds, which can be produced from pectin-rich food byproducts. In the present study, different technological means of POS production were explored to produce tailor-made POS mixtures starting from sugar beet pulp. The overall POS production process consisted of two steps: the extraction of pectin and the hydrolysis of pectin to tailored POS by combined hydrolysis/fractionation approaches. Different extraction as well as hydrolysis and fractionation methodologies were applied. The obtained POS were characterized for their total galacturonic acid content and, at a deeper level, using a HILIC-ESI/MS methodology, for the POS structure and composition. The composition of POS fractions was studied as a function of the technology used to obtain them. Finally, the potential prebiotic properties of the POS mixtures obtained were thoroughly explored by several in vitro experiments aimed at detecting lactic acid bacteria (LAB) stimulation by POS fractions. Several fractions were very efficient in stimulation, in a species-dependent manner. The overall best fractions were in general those rich in arabinans having a low degree of polymerization, obtained from the enzymatic extraction of biomass and subsequent fractionation with low-medium molecular weight cut-off. Quite interestingly, no POS fraction was able to stimulate pathogenic E. coli strains. The data reported here clearly indicate the possibility to obtain diverse fractions with different prebiotic properties starting from the same biomass, and outline clear potential for POS obtained from sugar beet pulp with the appropriate technology to act as prebiotic compounds.

This study was to identify active pectin fragments of high in vitro anti-proliferation activities against human colon adenocarcinoma Caco-2 cells, using various pectin fragment preparations dominant with rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and galacturonic acid oligomers (GAOS). The samples were prepared from citrus high and low methoxy pectins (HPE and LPE) by endo-α-D-(1,4)-polygalacturonase action coupling with a green process as ultrafiltration (membrane molecular weight cut off  > 10k, 1k-10k, and < 1k, giving fragment preparations I, II, and III, respectively). It is indicated that, in the fragment preparations HPE-I, II, and III, the molecular fractions HPE-F1 (RG-I), HPE-F2 (RG-II), and HPE-F3(GAOS) showed an averaged M^w = 26.7k, 3.4k, and 0.3k, respectively, notably higher than their LPE counterparts. All fragment preparations exhibited concentration-dependent, inhibition effects on Caco-2 cells, where HPE-II (RG-II-domain) revealed an interesting inhibition effect, significantly higher than those did HPE-I (RG-I domain) and HPE-III (GAOS). The HPE-F2 (RG-II) was elucidated and found of IR = 88% at 1.0 mg/mL and half-inhibition concentration (IC⁵⁰) = 0.12 mg/mL, doubling the effect of HPE-F1 (RG-I) (IR = 45% at 1.0 mg/mL). Conclusively, citrus RG-II fragment showed a promisingly high anti-Caco-2 activity, significantly higher than did RG-I and GAOS, possibly attributed to its special branched structure and low molecular size.

Tomato fruit texture depends on histology and cell wall architecture, both under genetic and developmental controls. If ripening related cell wall modifications have been well documented with regard to softening, little is known about cell wall construction during early fruit development. Identification of key events and their kinetics with regard to tissue architecture and cell wall development can provide new insights on early phases of texture elaboration. In this study, changes in pectin and hemicellulose chemical characteristics and location were investigated in the pericarp tissue of tomato (Solanum lycopersicon var Levovil) at four stages of development (7, 14 and 21 day after anthesis (DPA) and mature green stages). Analysis of cell wall composition and polysaccharide structure revealed that both are continuously modified during fruit development. At early stages, the relative high rhamnose content in cell walls indicates a high synthesis of rhamnogalacturonan I next to homogalacturonan. Fine tuning of rhamnogalacturonan I side chains appears to occur from the cell expansion phase until prior to the mature green stage. Cell wall polysaccharide remodelling also concerns xyloglucans and (galacto)glucomannans, the major hemicelluloses in tomato cell walls. In situ localization of cell wall polysaccharides in pericarp tissue revealed non-ramified RG-I rich pectin and XyG at cellular junctions and in the middle lamella of young fruit. Blocks of non-methyl esterified homogalacturonan are detected as soon as 14 DPA in the mesocarp and remained restricted to cell corner and middle lamella whatever the stages. These results point to new questions about the role of pectin RGI and XyG in cell adhesion and its maintenance during cell expansion.

The use of polygalacturonase (PG) preparations in winemaking promotes the release of phenolic compounds. A PG from a new source, Aspergillus sojae mutant, was semi-purified and tested for grape mash maceration. Crude extract (CE), a commercial pectinase, and two high PG activity semi-purified preparations, F_I and F_II, were applied for maceration at PG activity of 3.5 U g^-1 of grape for 46 h. Enzyme-assisted maceration significantly (P < 0.05) increased the total phenolic content from 255.8 to 916.3 ± 5.2, 5732.9 ± 9.9, 563.4 ± 6.7 and 620.6 ± 18.4 mg L^-1 for CE, commercial pectinase, F_I and F_II, respectively. The content of individual phenolics such as gallic, protocatechuic, chlorogenic and p-coumaric acids was improved. Principal component and hierarchical clustering analyses suggested that CE has a better performance upon the release of phenols. Semi-purified preparations acted similar to commercial pectinase. These findings open an opportunity for the potential use of PG from the mutant strain as an alternative macerating enzyme.

One of the four pectin methylesterase types isolated from Citrus sinensis var. Valencia fruit was used to demethylesterify a model homogalacturonan (HG) to 30%, 50% and 70% degree of methylesterification (DM) at pH 4.5 and 7.0, respectively. Introduced demethylesterified blocks (DMBs) were released by a limited  endo-polygalacturonase (EPG) digestion, separated and quantified by HPAEC. Average DMB size (BS) and number of such blocks per molecule (BN) differed depending on final DM and reaction pH (P < 0.05). BS and BN were significantly higher in 30% DM HG than 50 and 70 DMs. pH 4.5 series showed significantly larger BS compared to pH 7.5 series (P < 0.01). Distribution of DMBs released by limited EPG digest was predicted by mathematical modeling and in silico modeled processive (degree of processivity = 10), multiple attack mode of action best explains the experimental block distributions. Absolute degree of blockiness (DB_abs) obtained from exhaustive   EPG digestions, displayed a linear relationship with DM regardless of reaction pH (P < 0.001). Significant correlation coefficients between BS, BN, DB_abs, and DM manifested the effectiveness of the block information gained from both EPG digestion to estimate DMB distribution pattern (P < 0.05). However, comparison of block distribution information of three isozymes revealed that difference in block pattern could be manifested by parameters from limited EPG digest (BS, BN ) but not by those from exhaustive digest (DB_abs). The results suggested the possibility to control BS and to customize specific population of demethylesterified pectin molecules using PME isozymes from Valencia orange.

Background: Unlike in abscission or dehiscence, fruit of kiwifruit Actinidia eriantha develop the ability for peel detachment when they are ripe and soft in the absence of a morphologically identifiable abscission zone. Two closely-related genotypes with contrasting detachment behaviour have been identified. The ‘good-peeling’ genotype has detachment with clean debonding of cells, and a peel tissue that does not tear. The ‘poor-peeling’ genotype has poor detachability, with cells that rupture upon debonding, and peel tissue that fragments easily. Results: Structural studies indicated that peel detachability in both genotypes occurred in the outer pericarp beneath the hypodermis. Immunolabelling showed differences in methylesterification of pectin, where the interface of labelling coincided with the location of detachment in the good-peeling genotype, whereas in the poor-peeling genotype, no such interface existed. This zone of difference in methylesterification was enhanced by differential cell wall changes between the peel and outer pericarp tissue. Although both genotypes expressed two polygalacturonase genes, no enzyme activity was detected in the good-peeling genotype, suggesting limited pectin breakdown, keeping cell walls strong without tearing or fragmentation of the peel and flesh upon detachment. Differences in location and amounts of wall-stiffening galactan in the peel of the good-peeling genotype possibly contributed to this phenotype. Hemicellulose-acting transglycosylases were more active in the good-peeling genotype, suggesting an influence on peel flexibility by remodelling their substrates during development of detachability. High xyloglucanase activity in the peel of the good-peeling genotype may contribute by having a strengthening effect on the cellulose-xyloglucan network. Conclusions: In fruit of A. eriantha, peel detachability is due to the establishment of a zone of discontinuity created by differential cell wall changes in peel and outer pericarp tissues that lead to changes in mechanical properties of the peel. During ripening, the peel becomes flexible and the cells continue to adhere strongly to each other, preventing breakage, whereas the underlying outer pericarp loses cell wall strength as softening proceeds. Together these results reveal a novel and interesting mechanism for enabling cell separation.

Dietary non-digestible polysaccharides (NDPs) might promote intestinal health via immuno-modulation. Immunomodulatory effects of NDP are most likely brought about by antigen processing cells such as macrophages that populate the intestine, although the mechanisms are still poorly understood. We validated the in vitro model of M1 and M2 macrophages to mimic the intestinal inflammatory and tolerant macrophages using literature and microarray-derived gene markers. All these markers were used to characterise the macrophage phenotype following NDP stimulation. This identified an alternative subset, termed M(NDP), which commonly modulated a set of 126 genes, involved in migration, metabolic processes, cell cycle, and inflammatory immune function. This gene-based analysis for macrophage subsets provides an additional tool to characterise NDP bioactivity for their in vivo potential.

It has long been accepted that differential radial thickening of guard cells plays an important role in the turgor-driven shape changes required for stomatal pore opening to occur. This textbook description derives from an original interpretation of structure rather than measurement of mechanical properties. Here we show, using atomic force microscopy, that although mature guard cells display a radial gradient of stiffness, this is not present in immature guard cells, yet young stomata show a normal opening response. Finite element modeling supports the experimental observation that radial stiffening plays a very limited role in stomatal opening. In addition, our analysis reveals an unexpected stiffening of the polar regions of the stomata complexes, both in Arabidopsis and other plants, suggesting a widespread occurrence. Combined experimental data (analysis of guard cell wall epitopes and treatment of tissue with cell wall digesting enzymes, coupled with bioassay of guard cell function) plus modeling lead us to propose that polar stiffening reflects a mechanical, pectin-based pinning down of the guard cell ends, which restricts increase of stomatal complex length during opening. This is predicted to lead to an improved response sensitivity of stomatal aperture movement with respect to change of turgor pressure. Our results provide new insight into the mechanics of stomatal function, both negating an established view of the importance of radial thickening and providing evidence for a significant role for polar stiffening. Improved stomatal performance via altered cell-wall-mediated mechanics is likely to be of evolutionary and agronomic significance.

In the plant cell wall, boron links two pectic domain rhamnogalacturonan II (RG-II) chains together to form a dimer and thus contributes to the reinforcement of cell adhesion. We studied the mur1-1 mutant of Arabidopsis thaliana which has lost the ability to form GDP-fucose in the shoots and show that the extent of RG-II cross-linking is reduced in the lignified stem of this mutant. Surprisingly, MUR1 mutation induced an enrichment of resistant interunit bonds in lignin and triggered the overexpression of many genes involved in lignified tissue formation and in jasmonic acid signaling. The defect in GDP-fucose synthesis induced a loss of cell adhesion at the interface between stele and cortex, as well as between interfascicular fibers. This led to the formation of regenerative xylem, where tissue detachment occurred, and underlined a loss of resistance to mechanical forces. Similar observations were also made on bor1-3 mutant stems which are altered in boron xylem loading, leading us to suggest that diminished RG-II dimerization is responsible for regenerative xylem formation.

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 demethylesterify 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 demethylesterification 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<sup-) and number of average sized demethylesterified blocks per molecule (BN^-) were estimated. BS^- and BN^- as well as DB/DBabsdiffered 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 CpL PME 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^- and engineering a population of demethylesterified pectin molecules with specified demethylesterified BS^- and functional properties.

The major polysaccharides present in the primary and secondary walls surrounding plant cells have been well characterized. However, our knowledge of the early stages of secondary wall formation is limited. To address this, cell walls were isolated from differentiating xylem vessel elements of tobacco bright yellow-2 (BY-2) cells induced by VASCULAR-RELATED NAC-DOMAIN7 (VND7). The walls of induced VND7-VP16-GR BY-2 cells consisted of cellulose, pectic polysaccharides, hemicelluloses, and lignin, and contained more xylan and cellulose compared with non-transformed BY-2 and uninduced VND7-VP16-GR BY-2 cells. A reducing end sequence of xylan containing rhamnose and galaturonic acid- residues is present in the walls of induced, uninduced, and non-transformed BY-2 cells. Glucuronic acid residues in xylan from walls of induced cells are O-methylated, while those of xylan in non-transformed BY-2 and uninduced cells are not. Our results show that xylan changes in chemical structure and amounts during the early stages of xylem differentiation.

An acidic polysaccharide, named as p-LBP, was isolated from Lycium barbarum L. by water extraction and purified by decoloration, ion exchange chromatography, dialysis and gel chromatography, successively. The primary structure analysis was determined by HPAEC-PAD, HPSEC, FT-IR, GC–MS, and NMR. The results showed p-LBP was a homogeneous heteropolysaccharide as a pectin molecule with an average molecular weight of 64 kDa p-LBP was an approximately 87 nm hollow sphere in 0.05 mol/L sodium sulfate solution determined by HPSEC-MALLS, DLS and TEM. A discussion of degradation patterns gave the detailed structural information of p-LBP. Therefore, the results from degraded fragments elucidated that the backbone of p-LBP was formed by → 4-7alpha;-GalpA-(1 →, repeatedly. Partial region was connected by → 4-α-GalpA-(1 → and → 2-α-Rhap-(1 →, alternatively. On the C-4 of partial → 2-α-Rhap-(1 → residues existed branches forming by → 4-β-Galp-(1 →, → 3-β-Galp-(1 → or → 5-α-Araf-(1 →, while on the C-6 of partial → 3-β-Galp-(1 → residues existed secondary branches forming by terminal-α-Araf, terminal-β-Galp or → 3-α-Araf-(1 →.

Dimerization of rhamnogalacturonan-II (RG-II) via boron cross-links contributes to the assembly and biophysical properties of the cell wall. Pure RG-II is efficiently dimerized by boric acid B(OH)₃ in vitro only if nonbiological agents for example Pb²⁺ are added. By contrast, newly synthesized RG-II domains dimerize very rapidly in vivo. We investigated biological agents that might enable this. We tested for three such agents: novel enzymes, borate-transferring ligands and cationic ‘chaperones’ that facilitate the close approach of two polyanionic RG-II molecules. Dimerization was monitored electrophoretically. Parsley shoot cell-wall enzymes did not affect RG-II dimerization in vitro. Borate-binding ligands (apiose, dehydroascorbic acid, alditols) and small organic cations (including polyamines) also lacked consistent effects. Polylysine bound permanently to RG-II, precluding electrophoretic analysis. However, another polycation, polyhistidine, strongly promoted RG-II dimerization by B(OH)₃ without irreversible polyhistidine–RG-II complexation. Likewise, partially purified spinach extensins (histidine/lysine-rich cationic glycoproteins), strongly promoted RG-II dimerization by B(OH)₃ in vitro. Thus certain polycations, including polyhistidine and wall glycoproteins, can chaperone RG-II, manoeuvring this polyanionic polysaccharide domain such that boron-bridging is favoured. These chaperones dissociate from RG-II after facilitating its dimerization, indicating that they act catalytically rather than stoichiometrically. We propose a natural role for extensin–RG-II interaction in steering cell-wall assembly.

Water-soluble polysaccharides from 51 batches of fruits of L. barbarum (wolfberry) in China were investigated and compared using saccharide mapping, partial acid hydrolysis, single and composite enzymatic digestion, followed by polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) analysis and high performance thin layer chromatography (HPTLC) analysis, respectively. Results showed that multiple PACE and HPTLC fingerprints of partial acid and enzymatic hydrolysates of polysaccharides from L. barbarum in China were similar, respectively. In addition, results indicated that β-1,3-glucosidic, α-1,4-galactosiduronic and α-1,5-arabinosidic linkages existed in polysaccharides from L. barbarum collected in China, and the similarity of polysaccharides in L. barbarum collected from different regions of China was pretty high, which are helpful for the improvement of the performance of polysaccharides from L. barbarum in functional/health foods area. Furthermore, polysaccharides from Panax notoginseng, Angelica sinensis, and Astragalus membranaceus var. mongholicus were successfully distinguished from those of L. barbarum based on their PACE fingerprints. These results were beneficial to improve the quality control of polysaccharides from L. barabrum and their products, which suggested that saccharide mapping based on PACE and HPTLC analysis could be a routine approach for quality control of polysaccharides.

The natural consistency of processed tomato products arises from cell wall particles and the interactions between them. In this study, ion exchange resins were used to investigate these interactions. Two types of resins were used, a hydrogen form cation exchange resin and an anion resin in the hydroxide form. Serum phase of tomato suspensions were treated with either a cationic or an anionic resin to exchange various ionic compounds with hydrogen and hydroxide ion respectively. The treated serum was then reconstituted to the tomato pulp and the suspension was re-suspended with shear. The linear storage modulus varied with the different types of resin treatment. Samples treated with the anion exchange resin resulted in a higher modulus than the untreated tomato puree and the puree treated with the cation resin. Effect of the resins was dependent on the concentration of resin used. The anion treated sample resulted in a network formation which was quite sensitive to pH and was attributed to long range electrostatic interactions caused by protein–pectin interactions. Using Infrared spectroscopy the conformational changes in the protein structure as a result of resin treatment was detected by analyzing the amide-I and amide-II regions.