Pectin Components Research Articles

Heterologous expression of a Penicillium purpurogenum exo-arabinanase in Pichia pastoris and its biochemical characterization

Arabinan is a component of pectin, which is one of the polysaccharides present in lignocelluose. The enzymes degrading the main chain of arabinan are the endo- (EC 3.2.1.99) and exo-arabinanases (3.2.1.-). Only three exo-arabinanases have been biochemically characterized; they belong to glycosyl hydrolase family 93. In this work, the cDNA of an exo-arabinanase (Arap2) from Penicillium purpurogenum has been heterologously expressed in Pichia pastoris. The gene is 1310 bp long, has three introns and codes for a protein of 380 amino acid residues; the mature protein has a calculated molecular mass of 39 823 Da. The heterologously expressed Arap2 has a molecular mass in the range of 60–80 kDa due to heterogeneous glycosylation. The enzyme is active on debranched arabinan with optimum pH of 4–5.5 and optimal temperature of 40 °C, and has an exo-type action mode, releasing arabinobiose from its substrates. The expression profile of arap2 in corncob and sugar beet pulp follows a different pattern and is not related to the presence of arabinan. This is the first exo-arabinanase studied from P. purpurogenum and the first expressed in yeast. The availability of heterologous Arap2 may be useful for biotechnological applications requiring acidic conditions.

A phage display-selected peptide inhibitor of Agrobacterium vitis polygalacturonase.

Agrobacterium vitis, the causal agent of crown gall of grapevine, is a threat to viticulture worldwide. A major virulence factor of this pathogen is polygalacturonase, an enzyme that degrades pectin components of the xylem cell wall. A single gene encodes for the polygalacturonase gene. Disruption of the polygalacturonase gene results in a mutant that is less pathogenic and produces significantly fewer root lesions on grapevines. Thus, the identification of peptides or proteins that could inhibit the activity of polygalacturonase could be part of a strategy for the protection of plants against this pathogen. A phage-displayed combinatorial peptide library was used to isolate peptides with a high binding affinity to A. vitis polygalacturonase. These peptides showed sequence similarity to regions of Oryza sativa (EMS66324, Japonica) and Triticum urartu (NP_001054402, wild wheat) polygalacturonase-inhibiting proteins (PGIPs). Furthermore, these panning experiments identified a peptide, SVTIHHLGGGS, which was able to reduce A. vitis polygalacturonase activity by 35% in vitro. Truncation studies showed that the IHHL motif alone is sufficient to inhibit A. vitis polygalacturonase activity.

Pyrolysis and gasification of typical components in wastes with macro-TGA

The pyrolysis and gasification of typical components of solid waste, cellulose, hemicellulose, lignin, pectin, starch, polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC) and poly(ethylene terephthalate) (PET) were performed and compared in a macro thermogravimetric analyzer (macro-TGA). Three model biomasses, poplar stem, orange peel and Chinese cabbage, were applied to pyrolysis and gasification simulation by their components based on TG curves. Compared to those from TGA, peaks temperature of the differential thermogravimetric (DTG) curves of each samples pyrolysis on macro-TGA delayed 30–55°C due to heat transferring effect. CO2 promoted the thermal decomposition of hemicellulose, lignin, starch, pectin and model biomasses significantly by Boudouard reaction, and enhanced slightly the decomposition of PET. The activation energy (AE) of biomass components pyrolysis on macro-TGA was 167–197kJ/mol, while that of plastic samples was 185–235kJ/mol. The activation energy of 351–377kJ/mol was corresponding to the Boudouard reaction in CO2 gasification. All overlap ratios in pseudo-components simulation were higher than 0.98 to indicate that pseudo-components model could be applied to both pyrolysis and CO2 gasification, and the mass fractions of components derived from pyrolysis and gasification were slightly different but not brought in obvious difference in simulating curves when they were applied across.

Enzymatic Solubilisation and Degradation of Soybean Fibre Demonstrated by Viscosity, Fibre Analysis and Microscopy

The effect of a commercial multienzyme product obtained by fermentation from Aspergillus aculeatus on soybean and soybean meal was investigated using viscosity measurements, dietary fibre component analysis and different microscopy techniques utilizing histochemical dyes and antibody labelling. The results obtained demonstrated a strong viscosity reducing effect of the enzyme preparation on soluble galactomannan and xyloglucan polysaccharides and in addition non-starch polysaccharide analysis demonstrated a notable solubilisation of all polysaccharide constituents. The degradation of these components as native integral parts of cell walls upon exposure to the enzyme was visualized with microscopy. Two histochemical dyes, coriphosphine O and alcian blue were successfully used to follow pectin solubilisation after enzyme treatment. Commercial antibodies recognizing specific components of pectin and hemicellulose components of soybean cell wall were also used to visualize several enzyme activities in the commercial enzyme preparation The challenges of using commercial antibodies elicited from a given plant source to detect similar epitiopes on another plant source are also discussed. Non-starch polysaccharide analysis of the insoluble dietary fibre constituents before and after enzyme treatment corroborated the visualized mode of action demonstrated by microscopy. The combination of techniques provided visual and quantitative measurements of the solubilisation and degradation of hemicellulose pectic soybean cell wall components as part of the undesirable antinutrients in animal feed.

Tuning of pectin methylesterification: consequences for cell wall biomechanics and development.

Recent publications have increased our knowledge of how pectin composition and the degree of homogalacturonan methylesterification impact the biochemical and biomechanical properties of plant cell walls, plant development, and plants' interactions with their abiotic and biotic environments. Experimental observations have shown that the relationships between the DM, the pattern of de-methylesterificaton, its effect on cell wall elasticity, other biomechanical parameters, and growth are not straightforward. Working towards a detailed understanding of these relationships at single cell resolution is one of the big tasks of pectin research. Pectins are highly complex polysaccharides abundant in plant primary cell walls. New analytical and microscopy techniques are revealing the composition and mechanical properties of the cell wall and increasing our knowledge on the topic. Progress in plant physiological research supports a link between cell wall pectin modifications and plant development and interactions with the environment. Homogalacturonan pectins, which are major components of the primary cell wall, have a potential for modifications such as methylesterification, as well as an ability to form cross-linked structures with divalent cations. This contributes to changing the mechanical properties of the cell wall. This review aims to give a comprehensive overview of the pectin component homogalacturonan, including its synthesis, modification, regulation and role in the plant cell wall.

ВСТАНОВЛЕННЯ УМОВ СТАБІЛІЗАЦІЇ МАГНІТНИХ НАНОЧАСТИНОК У СКЛАДІ СИСТЕМ МАГНІТОКЕРОВАНОГО ТАРГЕТІНГУ ЛІКАРСЬКИХ РЕЧОВИН

ВСТАНОВЛЕННЯ УМОВ СТАБІЛІЗАЦІЇ МАГНІТНИХ НАНОЧАСТИНОК У СКЛАДІ СИСТЕМ МАГНІТОКЕРОВАНОГО ТАРГЕТІНГУ ЛІКАРСЬКИХ РЕЧОВИН

Rhamnogalacturonan I containing homogalacturonan inhibits colon cancer cell proliferation by decreasing ICAM1 expression

Pectin modified with pH, heat or enzymes, has previously been shown to exhibit anti-cancer activity. However, the structural requirements for modified pectin bioactivity have rarely been addressed. In this study several pectin extracts representing different structural components of pectin were assessed for effects against colon cancer cells. Rhamnogalacturonan I (RGI) extracts reduced proliferation of DLD1 and HCT116 colon cancer cells in a dose- and time-dependent manner. RGI reduced ICAM1 gene expression and siRNA-mediated knockdown of ICAM1 expression decreased cell proliferation providing a potential novel mechanism for the anti-cancer activity of pectin. Structural analysis of bioactive and non-bioactive RGIs suggested that a homogalacturonan component is maybe essential for the anti-proliferative activity, furthering the understanding of the structural requirements for pectin bioactivity.

Pyrolysis and simulation of typical components in wastes with macro-TGA

Abstract The pyrolysis of typical components of biomass waste, cellulose, hemicellulose, lignin, pectin and starch was performed and compared in a continuous-weighting fixed bed reactor acting as a macro thermogravimetric analyzer (TGA), namely macro-TGA. The mass loss and mass loss rate with time derived from macro-TGA looks similar to TG curve and differential thermogravimetric (DTG) curve from TGA. Compared to those from TGA, the DTG peak of every sample pyrolysis on macro-TGA delayed, and only one main peak appeared on hemicellulose with macro-TGA. The residue of different samples was in decreasing sequence as lignin > pectin > hemicellulose > starch > cellulose. Furthermore, the pseudo-components model based on the TG/macro-TG curves simulation was analyzed and compared. The overlap ratios between raw TG/macro-TG curves and simulation curves through different methods were all higher than 0.977 at the heating rate of 10 °C/min to suggest that biomass waste pyrolysis characteristics could be well simulated by its components.

Seasonal changes in chlorine and methoxyl content of leaves of deciduous trees and their impact on release of chloromethane and methanol at elevated temperatures

Environmental context Chloromethane is the most abundant naturally produced chlorine-containing organic compound, responsible for ~16% of chlorine-catalysed stratospheric ozone destruction. A significant source of this gas is emission from biomass by reaction between chloride ion and methoxyl groups of the biopolymers pectin and lignin. The seasonal changes in the chlorine and methoxyl pools observed in leaves of several deciduous tree species have implications for understanding chlorine volatilisation during biomass burning and estimation of the global chloromethane budget. Abstract Atmospheric chloromethane (CH3Cl) plays a role in the destruction of stratospheric ozone. Previous studies suggest an important source of this gas is emission from leaves and leaf litter at ambient and elevated (150–350°C) temperatures. In this study, the total chlorine and OCH3 content of leaves of the deciduous temperate tree species ash, beech, Norway maple and oak were measured throughout the 2004 and 2005 growing seasons. The total chlorine content increased with leaf age. The overall seasonal accumulation varied between five- and twenty-one fold, dependent on both year and species. Throughout the 2004 growing season, the OCH3 pool and the release of CH3Cl and methanol (CH3OH) from leaves of ash and Norway maple were monitored on heating to 350°C. The amounts of CH3Cl released increased linearly as leaf chlorine accumulated whereas emissions of CH3OH did not substantially change. Conversion of chlorine to CH3Cl was lower in the spring than during the summer and autumnal senescence period, ranging from 22 to 58%. No correlation was found between leaf OCH3 content and either CH3Cl or CH3OH release. The percentage conversion of OCH3 to the summed concentrations of CH3OH and CH3Cl ranged from 41 to 66%. The plant components pectin and lignin were identified as two major sources of the CH3 group in CH3Cl and CH3OH and emissions ceased when the OCH3 pool contributing the methyl moiety was exhausted (>350°C). These findings have implications for estimation of CH3Cl release during biomass burning and for our understanding of chlorine volatilisation during energy production from biomass.

Properties Characterization of Chemically Modified Hemp Hurds.

The effect of chemical treatment of hemp hurds slices in three solutions (EDTA (Ethylenediaminetetraacetic acid), NaOH and Ca(OH)2) on the properties of natural material was discussed in this paper. Changes in the morphology, chemical composition and structure as well as thermal stability of hemp hurds before and after their modification were investigated by using FTIR (Fourier transform infrared spectroscopy), XRD (X-ray powder diffraction analysis) and TG (thermogravimetry)/DSC (differential scanning calorimetry). Size exclusion chromatography (SEC) measurements were used for determination of degree of cellulose polymerization of hemp hurd samples. Chemical modification is related to the partial removal of non-cellulosic components of lignin, hemicellulose and pectin as well as waxes from the surface of hemp hurd slices. Another effect of the chemical treatment applied is connected with increasing the crystallinity index of cellulose determined by FTIR and XRD methods. Decrease in degree of cellulose polymerization and polydispersity index in chemically modified hemp hurds compared to the original sample was observed. Increase in thermal stability of treated hemp hurd was found. The most significant changes were observed in alkaline treated hemp hurds by NaOH.

Characterization of citrus pectin samples extracted under different conditions: influence of acid type and pH of extraction.

Pectin is a complex macromolecule, the fine structure of which is influenced by many factors. It is used as a gelling, thickening and emulsifying agent in a wide range of applications, from food to pharmaceutical products. Current industrial pectin extraction processes are based on fruit peel, a waste product from the juicing industry, in which thousands of tons of citrus are processed worldwide every year. This study examines how pectin components vary in relation to the plant source (orange, lemon, lime, grapefruit) and considers the influence of extraction conditions on the chemical and macromolecular characteristics of pectin samples. Citrus peel (orange, lemon, lime and grapefruit) from a commercial supplier was used as raw material. Pectin samples were obtained on a bulk plant scale (kilograms; harsh nitric acid, mild nitric acid and harsh oxalic acid extraction) and on a laboratory scale (grams; mild oxalic acid extraction). Pectin composition (acidic and neutral sugars) and physicochemical properties (molar mass and intrinsic viscosity) were determined. Oxalic acid extraction allowed the recovery of pectin samples of high molecular weight. Mild oxalic acid-extracted pectins were rich in long homogalacturonan stretches and contained rhamnogalacturonan I stretches with conserved side chains. Nitric acid-extracted pectins exhibited lower molecular weights and contained rhamnogalacturonan I stretches encompassing few and/or short side chains. Grapefruit pectin was found to have short side chains compared with orange, lime and lemon. Orange and grapefruit pectin samples were both particularly rich in rhamnogalacturonan I backbones. Structural, and hence macromolecular, variations within the different citrus pectin samples were mainly related to their rhamnogalacturonan I contents and integrity, and, to a lesser extent, to the length of their homogalacturonan domains.

Disease Association and Arthritogenic Potential of Circulating Antibodies against the α1,4-Polygalacturonic Acid Moiety

Much progress has been made in recent years on the diagnostic value, Ag specificity, and pathogenic roles of autoantibodies correlated to the development of rheumatoid arthritis (RA) in humans. However, carbohydrate Ag-specific autoantibodies that may also play important roles in RA have largely been ignored. In this article, we report that serum levels of Abs capable of recognizing α1,4-polygalacturonic acid [(PGA); major structural component of pectin] strongly correlate with RA in humans. The measurements of PGA-specific Abs (PGA-Abs) in sera are comparable to rheumatoid factors and anti-cyclic citrullinated peptide Abs as serological diagnostic markers for RA in terms of sensitivity and specificity. Immunohistochemical staining results indicate that the PGA-Abs selectively bound synovial membrane cells and chondrocytes in the joints of both humans and rabbits (but not rodents). Induction of PGA-Abs by s.c. immunization of rabbits with carrier protein-conjugated synthetic PGA led to severe inflammatory reactions (synovial hyperplasia, small vessel proliferation, and inflammatory cell infiltration) in the joints. Injection of affinity purified anti-PGA IgG into the synovial cavity of rabbits resulted in accumulation of proinflammatory cytokines such as TNF-α, IL-8, and IL-1β in synovial fluid, as well as local pathological damage. We conclude that the PGA-cross-reactive moiety represents a major autoantigen in the joints and can be targeted by autoantibodies capable of triggering arthritogenic responses in vivo.

СОДЕРЖАНИЕ НЕКОТОРЫХ ГРУПП СОЕДИНЕНИЙ У HEMEROCALLIS MINOR В УСЛОВИЯХ ИНТРОДУКЦИИ

В условиях лесостепной зоны Западной Сибири впервые установлено содержание сахаров, крахмала, сапонинов, аскорбиновой кислоты, пектинов, протопектинов, катехинов, дубильных веществ в надземных и подземных органах у Hemerocallis minor. Определено количественное содержание некоторых запасных и биологически активных веществ в листьях и корневищах.

Purification, crystallization and preliminary X-ray diffraction analysis of a novel keto-deoxy-D-galactarate (KDG) dehydratase from Agrobacterium tumefaciens.

D-galacturonic acid is the main component of pectin. It could be used to produce affordable renewable fuels, chemicals and materials through biotechnical conversion. Keto-deoxy-D-galactarate (KDG) dehydratase is an enzyme in the oxidative pathway of D-galacturonic acid in Agrobacterium tumefaciens (At). It converts 3-deoxy-2-keto-L-threo-hexarate to α-ketoglutaric semialdehyde. At KDG dehydratase was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 169.1, b = 117.8, c = 74.3 Å, β = 112.4° and an asymmetric unit of four monomers. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The three-dimensional structure of At KDG dehydratase will provide valuable information on the function of the enzyme and will allow it to be engineered for biorefinery-based applications.

Compositional Analysis of Chinese Water Chestnut (Eleocharis dulcis) Cell-Wall Material from Parenchyma, Epidermis, and Subepidermal Tissues

Chinese water chestnut (Eleocharis dulcis (Burman f.) Trin ex Henschel) is a corm consumed globally in Oriental-style cuisine. The corm consists of three main tissues, the epidermis, subepidermis, and parenchyma; the cell walls of which were analyzed for sugar, phenolic, and lignin content. Sugar content, measured by gas chromatography, was higher in the parenchyma cell walls (931 μg/mg) than in the subepidermis (775 μg/mg) or epidermis (685 μg/mg). The alkali-extractable phenolic content, measured by high-performance liquid chromatography, was greater in the epidermal (32.4 μg/mg) and subepidermal cell walls (21.7 μg/mg) than in the cell walls of the parenchyma (12.3 μg/mg). The proportion of diferulic acids was higher in the parenchyma. The Klason lignin content of epidermal and subepidermal cell walls was ~15%. Methylation analysis of Chinese water chestnut cell-wall polysaccharides identified xyloglucan as the predominant hemicellulose in the parenchyma for the first time, and also a significant pectin component, similar to other nongraminaceous monocots.

FRUIT SET AND EARLY BERRY DEVELOPMENT IN TWO GRAPEVINE CULTIVARS

The growth of the ovary/fruitlet of the beiry of two grapevine cultivars (‘Barbera’ and ‘Freisa’) was investigated at the karyological and histochemical level, in order to study the relationship between mitosis and cell-wall development and the synthesis of polysaccharides. The experiment started when flower buds appeared and continued throughout the formation of fruitlets until early July. In general, the peak of mitosis occurred a few days after fruit set, and the nucellar tissues were affected by the mi- totic processes longer than any other ovary tissues. The growth of the berry was mainly due to the mesocarp cell enlargement It seems that the main components of the cell-wall were pectins, which appeared to be increasing during the mitotic phase of tissue growth. Afterwards, stretching of cell-walls proceeded at a faster pace than pectin synthesis. Differences between the varieties were small but definite, indicating that they may be genotypical.

Substrate Dynamics in Enzyme Action: Rotations of Monosaccharide Subunits in the Binding Groove are Essential for Pectin Methylesterase Processivity

The dynamical behavior of biomacromolecules is a fundamental property regulating a large number of biological processes. Protein dynamics have been widely shown to play a role in enzyme catalysis; however, the interplay between substrate dynamics and enzymatic activity is less understood. We report insights into the role of dynamics of substrates in the enzymatic activity of PME from Erwinia chrysanthemi, a processive enzyme that catalyzes the hydrolysis of methylester groups from the galacturonic acid residues of homogalacturonan chains, the major component of pectin. Extensive molecular dynamics simulations of this PME in complex with decameric homogalacturonan chains possessing different degrees and patterns of methylesterification show how the carbohydrate substitution pattern governs the dynamics of the substrate in the enzyme’s binding cleft, such that substrate dynamics represent a key prerequisite for the PME biological activity. The analyses reveal that correlated rotations around glycosidic bonds of monosaccharide subunits at and immediately adjacent to the active site are a necessary step to ensure substrate processing. Moreover, only substrates with the optimal methylesterification pattern attain the correct dynamical behavior to facilitate processive catalysis. This investigation is one of the few reported examples of a process where the dynamics of a substrate are vitally important.

Pulping Properties of Bast Fibers of Paper Mulberry by Pre-steaming and 2-stage Cooking System

The traditional Hanji-making was confronted with lots of industrial disadvantages and economic problems, due to the original hand-made process. Recently, the studies on the automation of overall Hanji manufacturing process is carried out by applying the commercial chemical pulping method in order to expand industrial application or efficiency of non-wood fibrous materials. However, the application of commercial pulping methods to the bast tissues of paper mulberry leads to the chemical and mechanical deterioration of cellulosic fibers. In this study, the optimal cooking method using the bast parts of paper mulberry produced by an auto-scraping device was applied to minimize the damage of fiber strength for the paper yarn manufacture. The pre-steaming treatment and alkaline pulping systems were evaluated in removal efficiency of lignin and pectin materials within the bast tissue of paper mulberry. With the application of pre-steaming treatment and 2 stage pulping system using potassium carbonate and then sodium hydroxide, kappa values were decreased two times more in lignin removal than the single stage of pulping method. It was also identified from SEM images and ATR-FTIR spectra that the pectin components within cellular structure of bast tissue were easily removed and the debarked bast parts by a auto-scraping device were easily defiberized by 2-stage pulping sequence using potassium carbonate/sodium hydroxide pulping system.

Pectin Biosynthesis: GALS1 in Arabidopsis thaliana Is a β-1,4-Galactan β-1,4-Galactosyltransferase

β-1,4-Galactans are abundant polysaccharides in plant cell walls, which are generally found as side chains of rhamnogalacturonan I. Rhamnogalacturonan I is a major component of pectin with a backbone of alternating rhamnose and galacturonic acid residues and side chains that include α-1,5-arabinans, β-1,4-galactans, and arabinogalactans. Many enzymes are required to synthesize pectin, but few have been identified. Pectin is most abundant in primary walls of expanding cells, but β-1,4-galactan is relatively abundant in secondary walls, especially in tension wood that forms in response to mechanical stress. We investigated enzymes in glycosyltransferase family GT92, which has three members in Arabidopsis thaliana, which we designated GALACTAN SYNTHASE1, (GALS1), GALS2 and GALS3. Loss-of-function mutants in the corresponding genes had a decreased β-1,4-galactan content, and overexpression of GALS1 resulted in plants with 50% higher β-1,4-galactan content. The plants did not have an obvious growth phenotype. Heterologously expressed and affinity-purified GALS1 could transfer Gal residues from UDP-Gal onto β-1,4-galactopentaose. GALS1 specifically formed β-1,4-galactosyl linkages and could add successive β-1,4-galactosyl residues to the acceptor. These observations confirm the identity of the GT92 enzyme as β-1,4-galactan synthase. The identification of this enzyme could provide an important tool for engineering plants with improved bioenergy properties.

Evidence for rapid uptake of d-galacturonic acid in the yeast Saccharomyces cerevisiae by a channel-type transport system

d-Galacturonic acid is a major component of pectins but cannot be metabolized by Saccharomyces cerevisiae. It is assumed not to be taken up. We show that yeast displays surprisingly rapid low-affinity uptake of d-galacturonic acid, strongly increasing with decreasing extracellular pH and without saturation up to 1.5M. There was no intracellular concentration above the extracellular level and transport was reversible. Among more than 160 single and multiple deletion mutants in channels and transporters, no strain was affected in d-galacturonic acid uptake. The uptake was not inhibited by any compound tested as candidate competitive inhibitor, including d-glucuronic acid, which was also transported. The characteristics of d-galacturonic acid uptake are consistent with involvement of a channel-type system, probably encoded by multiple genes.