Degree Of Methylesterification Research Articles

Biochemical Characterization, Thermal Stability, and Partial Sequence of a Novel Exo-Polygalacturonase from the Thermophilic Fungus Rhizomucor pusillus A13.36 Obtained by Submerged Cultivation.

This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45°C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5–47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61°C. It presents thermal stability between 30 and 60°C, has 70% activation in the presence of Ca2+, and was tested using citrus pectin with a degree of methyl esterification (DE) of 26%. E a(d) for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and ΔG (d) values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with an exo-pectinase from Aspergillus fumigatus. Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot.

Alkaline phosphatase immobilization on spherical pectin gel particles

Spherical gel particles from apple pectin and amidated citrus pectin with a low degree of methyl esterification were obtained by ionotropic gelation in the presence of calcium ions. A commercial preparation of alkaline phosphatase from bovine intestinal mucosa was used for enzyme immobilization by physical adsorption on pectin particles and by incorporation into a hydrogel. Alkaline phosphatase immobilized on pectin gels retained 21–55% of the initial activity. Release of the immobilized enzyme from the gel particles into solutions modeling the physiological conditions in different parts of the gastrointestinal tract was investigated. Alkaline phosphatase desorbed from pectin particles was shown to dephosphorylate bacterial lipopolysaccharide.

Mechanistic insight into common bean pectic polysaccharide changes during storage, soaking and thermal treatment in relation to the hard-to-cook defect

Different mechanisms responsible for the development of the hard-to-cook defect in common beans during storage, their soaking behavior and softening during thermal treatment have been previously suggested. However, these mechanisms have not been sufficiently confirmed by direct molecular evidence. This research aimed at gaining a detailed mechanistic insight into changes occurring in Canadian wonder bean pectic polysaccharides during storage, soaking and/or thermal treatment in different brine solutions in relation to the development and manifestation of the hard-to-cook (HTC) defect. Both fresh or easy-to-cook (ETC) and stored (HTC) bean samples were either soaked or soaked and thermally treated in demineralized water, solutions of Na2CO3 and CaCl2 salts followed by extraction of cell wall materials. Pectic polysaccharide properties examined included sugar composition, degree of methylesterification (DM), extractability and molar mass (MM). The DM of pectin from ETC and HTC beans was similar but low (<50%). Upon (pre)treatment in a Na2CO3 solution, solubilization of pectic polysaccharides, especially the strongly bound chelator- (CEP) and Na2CO3- (NEP) extractable pectins was enhanced leading to increased amounts of water extractable pectin (WEP). Also, there was a decrease in high MM polymers paralleled by an increase in β-elimination degradation products. These observations are in line with the fast cooking behavior of beans (pre)treated in a Na2CO3 solution. In contrast, (pre)treatment in a CaCl2 solution hindered softening leading to the failure of the beans to cook. The beans (pre)treated in a CaCl2 solution showed increased high MM polymers and lack of cell wall separation. Therefore, it can be inferred that development of the hard-to-cook defect in Canadian wonder beans during storage and its manifestation during soaking and subsequent thermal treatment is largely reflected by the pectic polysaccharide properties in line with the pectin hypothesis. Our data suggest the release of Ca++ leading to pectin cross-linking and the increase or decrease of β-elimination depolymerization. However, the relatively high amounts of neutral sugars and strongly bound NEP in HTC seeds do not allow to rule out the possible existence of non-Ca++ based pectin cross-linking.

Modifications of cell wall pectin in tomato cell suspension in response to cadmium and zinc

Retention of metal cations by the cell wall is a common process found in plants in response to stress induced by the presence of trace metals (TMs). In this study conducted on a tomato cell suspension culture, cadmium (Cd) and zinc (Zn) were added to the medium at maximal concentrations of 0.5 and 2 mM, respectively. We showed that around 50 % of Zn or Cd was confined into the cell wall of tomato cells. Besides, their accumulation in the cell wall increased with the exogenous concentration in the culture medium. Characterization of cell wall pectins showed a decrease in the highly methylesterified pectin fraction whereas the weakly methylesterified pectin remained stable in response to Cd. Moreover, a significant increase in the degree of methylesterification was observed in both fractions. This was probably associated to the reduced pectin methylesterase (PME) activity in the treated cells. Furthermore, linked to a reduction of pectin content we showed a reduced expression of the galacturonosyltransferase QUA1 gene whereas PME1 expression remained unchanged. Taking together, these data strongly suggest that pectin biosynthesis and its modification in the cell wall are strongly regulated in response to TM exposure in tomato cells.

Zooming in: Structural Investigations of Rheologically Characterized Hydrogen-Bonded Low-Methoxyl Pectin Networks.

Self-assembled hydrogen-bonded networks of the polysaccharide pectin, a mechanically functional component of plant cell walls, have been of recent interest as biomimetic exemplars of physical gels, and the microrheological and strain-stiffening behaviors have been previously investigated. Despite this detailed rheological characterization of preformed gels, little is known about the fundamental arrangement of the polymers into cross-linking junction zones, the size of these bonded regions, and the resultant network architecture in these hydrogen-bonded materials, especially in contrast to the plethora of such information available for their well-known calcium-assembled counterparts. In this work, in concert with pertinent rheological measurements, an in-depth structural study of the hydrogen-bond-mediated gelation of pectins is provided. Gels were realized by using glucona-delta-lactone to decrease the pH of solutions of pectic polymers that had a (blockwise) low degree of methylesterification. Small-angle X-ray scattering and transmission electron microscopy were utilized to access structural information on length scales on the order of nanometers to hundreds of nanometers, while complementary mechanical properties were measured predominantly using small amplitude oscillatory shear rheology.

Tuning of Pectin Methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.

Pectins esterification in the apoplast of aluminum-treated pea root nodules

Aiming to elucidate the possible involvement of pectins in aluminum-mediated growth inhibition the distribution of pectins in the apoplast of root nodules was investigated. Experiments were performed on the pea (Pisum sativum L.) root nodules treated with aluminum (50μM AlCl3, for 2 or 24h). For histochemical acidic pectin localization we used ruthenium red staining. Immunolabeling techniques with monoclonal antibodies specific to high methyl-esterified pectin (JIM7), low methyl-esterified pectin (JIM5) and calcium cross-linked pectin (2F4) were used to re-examine the pattern of pectin esterification and distribution. After immunolabeling the samples were observed using a fluorescent and transmission electron microscope.Ruthenium red staining showed that acid pectin content increased in the apoplast of Al-treated nodules and immunolocalization of pectin epitopes revealed that the fraction of de-esterified pectins increased significantly under Al stress. JIM5 and 2F4 epitopes were located on the inner surface of the primary cell wall with higher intensity at cell corners lining the intercellular spaces and at infection threads (ITs) walls. By contrast, JIM 7 labels all walls uniformly throughout the nodule. In the presence of Al, the increase of JIM5 and 2F4 labeling in thick plant and IT walls, together with a decrease of JIM7 labeling was observed.These results indicate a specific role for pectin de-esterification in the process of wall thickening and growth inhibition. In particular, Al-dependent increase in pectin content and their low methyl esterification degree correlate with wall thickness and higher rigidity, and in this way, may affect IT and nodules growth.

Microscopic evidence for Ca2+ mediated pectin–pectin interactions in carrot-based suspensions

This study explored the use of fluorescently labeled pectin to obtain evidence for Ca2+ mediated pectin–pectin interactions in situ. Specifically, carrots were either blanched at low temperature (LTB) or blanched at high temperature (HTB) to activate or inactivate endogenous pectin methylesterase, respectively. Consequently, pectin in tissue particles of LTB and HTB carrots exhibited low degree of methylesterification (DM) and high DM, respectively. Pectin present in the LTB carrot serum exhibited a lower DM, was more branched, and showed a higher molar mass compared to HTB carrot serum pectin. Ca2+ mediated pectin–pectin interactions were influenced by serum pectin molecular structure, increased with increasing pH and Ca2+ concentration, and decreasing DM. Presence of more linear pectin in the serum created a competition, leading to less intense interactions between labeled pectin and pectin at tissue particle surfaces. Generally, the most intense Ca2+ mediated pectin–pectin interactions were observed for pectin of LTB carrot particles.

A multivariate approach for high throughput pectin profiling by combining glycan microarrays with monoclonal antibodies

Pectin–one of the most complex biomacromolecules in nature has been extensively studied using various techniques. This has been done so in an attempt to understand the chemical composition and conformation of pectin, whilst discovering and optimising new industrial applications of the polymer. For the last decade the emergence of glycan microarray technology has led to a growing capacity of acquiring simultaneous measurements related to various carbohydrate characteristics while generating large collections of data. Here we used a multivariate analysis approach in order to analyse a set of 359 pectin samples probed with 14 different monoclonal antibodies (mAbs). Principal component analysis (PCA) and partial least squares (PLS) regression were utilised to obtain the most optimal qualitative and quantitative information from the spotted microarrays. The potential use of microarray technology combined with chemometrics for the accurate determination of degree of methyl-esterification (DM) and degree of blockiness (DB) was assessed.

Pectin homogalacturonans: Nanostructural characterization of methylesterified domains

Functionality of pectic hydrocolloids is largely dependent on the two major domains commonly found in their homogalacturonan regions, i.e., methylester protected domains (MPDs) and non methylesterified domains (NMDs). MPDs can participate in hydrogen bonding and hydrophobic interactions but unlike the NMDs they are incapable of calcium mediated cross-linking. MPDs also are inaccessible to endo polygalacturonase attack while NMDs are susceptible. We isolated and characterized MPDs contained within homogalacturonans that had been demethylesterified either by plant pectin methylesterases or chemically by base saponification. Molecular weight (∼9500–14,000 Da) and degree of methylesterification (DM; ∼63%–94%) of remnant MPDs were measured. Subsequently the MPDs were base saponified at 4 °C. The resulting demethylesterified oligomers were separated, visualized and quantified. Population parameters estimated were the average number of MPDs per pectin molecule, the sum of galacturonic acid units in MPDs per molecule, the average MPD size and the number of “average” size MPDs per molecule. Analysis of variance and Tukey's Multiple Comparison Test indicated significant differences in MPDs based on DM and the method of demethylesterification. Pearson's correlation coefficients were calculated for MPD parameters, previously published structural data from NMDs and rheological properties of the demethylesterified homogalacturonans from the enzyme treatments. Significant correlations were observed for DM, absolute degree of blockiness, G′, NMD and MPD parameters. When coupled with methods to characterize NMDs these techniques provide an opportunity to statistically describe both highly charged and more neutral domains in a population of pectin molecules.

Arabidopsis PME17 Activity can be Controlled by Pectin Methylesterase Inhibitor4

The degree of methylesterification (DM) of homogalacturonans (HGs), the main constituent of pectins in Arabidopsis thaliana, can be modified by pectin methylesterases (PMEs). Regulation of PME activity occurs through interaction with PME inhibitors (PMEIs) and subtilases (SBTs). Considering the size of the gene families encoding PMEs, PMEIs and SBTs, it is highly likely that specific pairs mediate localized changes in pectin structure with consequences on cell wall rheology and plant development. We previously reported that PME17, a group 2 PME expressed in root, could be processed by SBT3.5, a co-expressed subtilisin-like serine protease, to mediate changes in pectin properties and root growth. Here, we further report that a PMEI, PMEI4, is co-expressed with PME17 and is likely to regulate its activity. This sheds new light on the possible interplay of specific PMEs, PMEIs and SBTs in the fine-tuning of pectin structure.

The impact of rhamnogalacturonan-I side chain monosaccharides on the rheological properties of citrus pectin

Citrus peel is the preferred raw material for pectin production, mainly due to its high pectin content but also due to the gelling capabilities. Especially the high galacturonic acid content and the high degree of methyl esterification make it an attractive starting material for further modification. Until now the presence of rhamnogalacturonan-I (RG-I) in commercial pectin has received less attention due to its relatively low abundance. Using a gentle industrial extraction process we discovered a slightly higher composition in neutral sugars of RG-I. In particular, the arabinose concentration in mildly extracted pectin was higher compared to conventionally extracted pectin. The use of enzymes specific for either arabinan or galactan allowed assessing the implications of varying neutral sugar composition on the gelling capabilities of pectin. A comparison between untreated and debranched pectin revealed a significant decrease in both gelling temperature (up to 20 °C) and gel strength (up to 38%), following the specific reduction in neutral sugars concentration. Coil-overlap and entanglement effects were analysed to further understand the impact of RG-I side chains on polymer rheology.

Pectin characterisation in vegetable waste streams: A starting point for waste valorisation in the food industry

Vegetable waste streams, which are a major disposal issue in food industry, present a promising source of compounds which may be valorised because of their favourable functional features. In this context, the potential of five vegetable waste streams (rejected carrots, carrot steam peels, green beans cutting waste, leek cutting waste and celeriac steam peels) as a source for the extraction of pectin with interesting structural, and hence functional, properties was evaluated. Specifically, cell-wall components were extracted from the waste streams as alcohol-insoluble residue and subsequently fractionated into different (pectin) fractions based on their solubility. The pectic polysaccharides were characterised in terms of GalA content, neutral sugar content, linearity/branching, degree of methyl-esterification (DM), molar-mass distribution and the presence of bound protein. Pectin characterisation revealed considerable differences between the pectic polymers present in the investigated waste streams. For example, pectin in carrot steam peels showed a low DM, whereas the cutting waste of leek contained pectin with a very high DM. Furthermore, the level of protein bound to pectin was generally highest in carrot-derived waste streams. Depending on the intended pectin functionality, a deliberate choice for one of these vegetable waste streams as a source for pectin extraction can be made.

PECTIN METHYLESTERASE48 is involved in Arabidopsis pollen grain germination.

Germination of pollen grains is a crucial step in plant reproduction. However, the molecular mechanisms involved remain unclear. We investigated the role of PECTIN METHYLESTERASE48 (PME48), an enzyme implicated in the remodeling of pectins in Arabidopsis (Arabidopsis thaliana) pollen. A combination of functional genomics, gene expression, in vivo and in vitro pollen germination, immunolabeling, and biochemical analyses was used on wild-type and Atpme48 mutant plants. We showed that AtPME48 is specifically expressed in the male gametophyte and is the second most expressed PME in dry and imbibed pollen grains. Pollen grains from homozygous mutant lines displayed a significant delay in imbibition and germination in vitro and in vivo. Moreover, numerous pollen grains showed two tips emerging instead of one in the wild type. Immunolabeling and Fourier transform infrared analyses showed that the degree of methylesterification of the homogalacturonan was higher in pme48-/- pollen grains. In contrast, the PME activity was lower in pme48-/-, partly due to a reduction of PME48 activity revealed by zymogram. Interestingly, the wild-type phenotype was restored in pme48-/- with the optimum germination medium supplemented with 2.5 mm calcium chloride, suggesting that in the wild-type pollen, the weakly methylesterified homogalacturonan is a source of Ca(2+) necessary for pollen germination. Although pollen-specific PMEs are traditionally associated with pollen tube elongation, this study provides strong evidence that PME48 impacts the mechanical properties of the intine wall during maturation of the pollen grain, which, in turn, influences pollen grain germination.

FT-IR spectroscopy, a reliable method for routine analysis of the degree of methylesterification of pectin in different fruit- and vegetable-based matrices

The use of Fourier transform infrared (FT-IR) spectroscopy as a method for routine analysis of the degree of methylesterification (DM) of pectin was validated. The relationship between the ratio of the intensity of the peak at around 1740cm−1 (due to ester carbonyl group stretching) to the sum of the intensities of the peaks at around 1740 and 1630–1600cm−1 (due to carboxylate group stretching) and the DM of pectin in model and real systems was investigated. In model systems of low to medium DM with low added protein (⩽20%), accurate DM determinations were obtained without spectra deconvolution whilst for medium to high DM pectin with high added protein (⩾30%), peak deconvolution was vital. In real systems, good DM determinations were obtained without peak deconvolution except for broccoli-derived samples. Considering that broccoli is a protein-rich vegetable, better determinations of the DM were obtained using deconvoluted FT-IR spectra.

Effect of Molecular Weight Reduction, Acetylation and Esterification on the Emulsification Properties of Citrus Pectin

Citrus pectin was chemically and thermally modified in order to increase the hydrophobic character of the molecule and its adsorptivity to the oil–water interface. The degree of acetylation and methylesterification was increased and the molecular weight was reduced. The emulsion formation and stabilization properties of these modified pectins were evaluated by surface and interfacial tension measurements and emulsification experiments. For the production of emulsions, a high pressure homogenizer was used. The viscosity ratio between oil and emulsion phase was adjusted by varying the amount of added sucrose. Pectins with a higher degree of methylesterification (DE) decrease the interfacial tension significantly compared to the unmodified pectin. Pectins with increased degree of acetylation (DAc), however, show higher interfacial tension values. In emulsification experiments, pectins with a reduced molecular weight do neither significantly reduce droplet sizes nor improve emulsion stability. Pectins with increased DE or DAc reduce the Sauter mean diameter d3,2 of emulsions significantly and, in case of an DE increase, also show excellent long term stability. Their performance is also superior to sugar beet pectin under comparable experimental conditions.

Effect of ripening and heat processing on the physicochemical and rheological properties of pepper pectins

Water-, chelator-, and alkali-soluble pectins were isolated from raw and heat-processed Jalapeño peppers (green and red) and their physiochemical and rheological properties were determined. The yield, tristimulus color, degree of methyl esterification, monosaccharide composition, molecular weights distribution, and protein content depended on ripening and heat processing. The viscosity properties of pectins were independent of ripening. The water-soluble pectin was the most abundant pectin. Pectins from grilled peppers showed the lowest L* values. The alkali-soluble pectin showed the highest protein content. The content of xylose, rhamnose, and mannose in pectins was highly altered by tested factors. The degree of methyl esterification of pectins ranged from 26.8 to 91.6%. The peak Mw of the main fraction of tested pectins was sequentially reduced by ripening and heat processing. Pectins from raw peppers showed the best viscosity properties.

Rheological characterization of acid pectin samples in the absence and presence of monovalent ions

Pectins are traditionally divided into two groups, high methoxy and low methoxy. The groupings determine the charge of the pectin and the gelation mechanism. However, not as yet extensively studied is the impact on gelation of the distribution of the charges as characterized by an absolute degree of blockiness (DBabs). The aim of this study was to characterize rheologically the acid gelation of a pectin with a high DBabs and a degree of methyl esterification of ∼37%, in the absence and presence of monovalent ions. The results obtained suggest that a pectin with a blocky charge distribution at pH conditions close to or below the pKa exhibits weak gel-like properties at intermediate frequencies, despite the absence of a permanent network structure. The addition of monovalent ions changed the rheological behavior to resemble that of a strong gel whose properties depended on the type and concentration of the ions.

Structural alteration of cell wall pectins accompanies pea development in response to cold

Pea (Pisum sativum) cell wall metabolism in response to chilling was investigated in a frost-sensitive genotype ‘Terese’ and a frost-tolerant genotype ‘Champagne’. Cell walls isolated from stipules of cold acclimated and non-acclimated plants showed that cold temperatures induce changes in polymers containing xylose, arabinose, galactose and galacturonic acid residues. In the tolerant cultivar Champagne, acclimation is accompanied by increases in homogalacturonan, xylogalacturonan and highly branched Rhamnogalacturonan I with branched and unbranched (1→5)-α-arabinans and (1→4)-β-galactans. In contrast, the sensitive cultivar Terese accumulates substantial amounts of (1→4)-β-xylans and glucuronoxylan, but not the pectins. Greater JIM7 labeling was observed in Champagne compared to Terese, indicating that cold acclimation also induces an increase in the degree of methylesterification of pectins. Significant decrease in polygalacturonase activities in both genotypes were observed at the end of cold acclimation. These data indicate a role for esterified pectins in cold tolerance. The possible functions for pectins and their associated arabinans and galactans in cold acclimation are discussed.

Structure of an arabinogalactan from the edible tropical fruit tamarillo (Solanum betaceum) and its antinociceptive activity.

A structural characterization of polysaccharides obtained by aqueous extraction of ripe pulp of the edible exotic tropical fruit named tamarillo (Solanum betaceum) was carried out. After fractionation by freeze-thaw and α-amylase treatments, a fraction containing a mixture of highly-methoxylated homogalacturonan and of arabinogalactan was obtained. A degree of methylesterification (DE) of 71% and a degree of acetylation (DA) of 1.3% was determined by (1)H NMR spectroscopy and spectrophotometric quantification, respectively. A type I arabinogalactan was purified via Fehling precipitation and ultrafiltration through 50 kDa (cut-off) membrane. Its chemical structure was performed by sugar composition, HPSEC, methylation, carboxy-reduction and (13)C NMR spectroscopy analysis. Intraperitoneal administration of the arabinogalactan did not reduce the nociception induced by intraplantar injection of 2.5% formalin in mice, but significantly reduced the number of abdominal constrictions induced by 0.6% acetic acid, indicating that fraction has an antinociceptive effect on the visceral inflammatory pain model.