Comprehensive Microarray Polymer Profiling Research Articles

Cell wall polysaccharide and glycoprotein content tracks growth-form diversity and an aridity gradient in the leaf-succulent genus Crassula.

Cell wall traits are believed to be a key component of the succulent syndrome, an adaptive syndrome to drought, yet the variability of such traits remains largely unknown. In this study, we surveyed the leaf polysaccharide and glycoprotein composition in a wide sampling of Crassula species that occur naturally along an aridity gradient in southern Africa, and we interpreted its adaptive significance in relation to growth form and arid adaptation. To study the glycomic diversity, we sampled leaf material from 56 Crassula taxa and performed comprehensive microarray polymer profiling to obtain the relative content of cell wall polysaccharides and glycoproteins. This analysis was complemented by the determination of monosaccharide composition and immunolocalization in leaf sections using glycan-targeting antibodies. We found that compact and non-compact Crassula species occupy distinct phenotypic spaces in terms of leaf glycomics, particularly in regard to rhamnogalacturonan I, its arabinan side chains, and arabinogalactan proteins (AGPs). Moreover, these cell wall components also correlated positively with increasing aridity, which suggests that they are likely advantageous in terms of arid adaptation. These differences point to compact Crassula species having more elastic cell walls with plasticizing properties, which can be interpreted as an adaptation toward increased drought resistance. Furthermore, we report an intracellular pool of AGPs associated with oil bodies and calcium oxalate crystals, which could be a peculiarity of Crassula and could be linked to increased drought resistance. Our results indicate that glycomics may be underlying arid adaptation and drought resistance in succulent plants.

Are cell wall traits a component of the succulent syndrome?

Succulence is an adaptation to low water availability characterised by the presence of water-storage tissues that alleviate water stress under low water availability. The succulent syndrome has evolved convergently in over 80 plant families and is associated with anatomical, physiological and biochemical traits. Despite the alleged importance of cell wall traits in drought responses, their significance in the succulent syndrome has long been overlooked. Here, by analyzing published pressure-volume curves, we show that elastic adjustment, whereby plants change cell wall elasticity, is uniquely beneficial to succulents for avoiding turgor loss. In addition, we used comprehensive microarray polymer profiling (CoMPP) to assess the biochemical composition of cell walls in leaves. Across phylogenetically diverse species, we uncover several differences in cell wall biochemistry between succulent and non-succulent leaves, pointing to the existence of a 'succulent glycome'. We also highlight the glycomic diversity among succulent plants, with some glycomic features being restricted to certain succulent lineages. In conclusion, we suggest that cell wall biomechanics and biochemistry should be considered among the characteristic traits that make up the succulent syndrome.

Focus on the relationships between the cell wall composition in the extraction of anthocyanins and tannins from grape berries

Concentrations of anthocyanins and tannins after extraction from berries in wines and from skin macerations in model solutions have been studied for two grape varieties, two maturation levels and two vintages berries. Characterization of the cell wall polysaccharides has also been performed, the classical method based on the analysis of the neutral sugars after depolymerization being completed by a comprehensive microarray polymer profiling (CoMPP). Extraction was lower in model solutions than in wines, with the same ranking: non acylated anthocyanins> tannins > p-coumaroylated anthocyanins. The polysaccharidic composition suggested a role of homogalacturonans, rhamnogalacturonans and extensins in the extraction process. A global explanation of the interactions between anthocyanins, tannins and polysaccharides is proposed.

Combined high-throughput and fractionation approaches reveal changes of polysaccharides in blueberry skin cell walls during fermentation for wine production

There have been rare reports about the structure/composition of polymers in blueberry skin and their changes during fermentation for wine production. In this study, the compositional changes occurring in blueberry skin during fermentation were tracked by a combination of cell wall analysis techniques including infra-red spectroscopy, monosaccharide analysis, and comprehensive microarray polymer profiling (CoMPP). The cross-corroborating data revealed that blueberry skin cell wall is particularly rich in xyloglucan. Chemical fractionation analysis indicated that the KOH soluble fraction is a dominant fraction in fermented blueberry skin. Interestingly, the KOH soluble fraction contained abundant epitopes associated with pectin branch chains, indicating tight binding of some enzyme-resistant pectin polymers to hemicellulose. This study provides important implications for the development of effective strategies to extract beneficial substances (such as aromatics, tannins and pigments) from berry tissues during processing.

Commercial Yeast Strains Expressing Polygalacturonase and Glucanase Unravel the Cell Walls of Chardonnay Grape Pomace.

Simple SummaryGrape skins, usually discarded during wine making, are a valuable source of cellulose (20–50%), hemicelluloses (15–20%), lignin (17–30%) and other compounds, e.g., polyphenols, which can be used as biomaterials in the manufacturing of a variety of new products, such as bioethanol or pharmaceutical products. However, to obtain these biomaterials, the complex polysaccharides of the grape cell walls must be broken down into smaller molecules to allow the extraction of compounds. The degradation process is often performed enzymatically or hydrothermally. Microorganisms that produce the required enzymes while using this waste product as a growth medium can have interesting economic advantages. Here, we created two genetically engineered wine yeast strains that produce grape cell wall degrading enzymes. These yeasts, when grown on grape pomace, induced enzymatic structural changes to the grape cell walls. A collection of antibodies binding to the different cell wall molecules were used to monitor the impact on the cell wall structure of the enzymes, confirming increased extractability of key cell wall polymers when relatively low levels of enzymes are present, illustrating the potential to develop and optimise yeast for grape waste valorisation applications.Industrial wine yeast strains expressing hydrolytic enzymes were fermented on Chardonnay pomace and were shown to unravel the cell walls of the berry tissues according to the enzyme activities. The yeasts produced a native endo-polygalacturonase (Saccharomyces cerevisiae × Saccharomyces paradoxus hybrid, named PR7) and/or a recombinant endo-glucanase (S. cerevisiae strains named VIN13 END1 and PR7 END1). The impact of the enzymes during the fermentations was evaluated by directly studying the cell wall changes in the berry tissues using a Comprehensive Microarray Polymer Profiling technique. By the end of the fermentation, the endo-glucanase did not substantially modify the berry tissue cell walls, whereas the endo-polygalacturonase removed some homogalacturonan. The recombinant yeast strain producing both enzymes (PR7 END1) unravelled the cell walls more fully, enabling polymers, such as rhamnogalacturonan-I, β-1,4-D-galactan and α-1,5-L-arabinan, as well as cell wall proteins to be extracted in a pectin solvent. This enzyme synergism led to the enrichment of rhamnogalacturonan-type polymers in the subsequent NaOH fractions. This study illustrated the potential utilisation of a recombinant yeast in pomace valorisation processes and simulated consolidated bioprocessing. Furthermore, the cell wall profiling techniques were confirmed as valuable tools to evaluate and optimise enzyme producing yeasts for grape and plant cell wall degradation.

Overexpression of UDP-sugar pyrophosphorylase leads to higher sensitivity towards galactose, providing new insights into the mechanisms of galactose toxicity in plants.

SUMMARYGalactose toxicity (Gal‐Tox) is a widespread phenomenon ranging from Escherichia coli to mammals and plants. In plants, the predominant pathway for the conversion of galactose into UDP‐galactose (UDP‐Gal) and UDP‐glucose is catalyzed by the enzymes galactokinase, UDP‐sugar pyrophosphorylase (USP) and UDP‐galactose 4‐epimerase. Galactose is a major component of cell wall polymers, glycolipids and glycoproteins; therefore, it becomes surprising that exogenous addition of galactose leads to drastic root phenotypes including cessation of primary root growth and induction of lateral root formation. Currently, little is known about galactose‐mediated toxicity in plants. In this study, we investigated the role of galactose‐containing metabolites like galactose‐1‐phosphate (Gal‐1P) and UDP‐Gal in Gal‐Tox. Recently published data from mouse models suggest that a reduction of the Gal‐1P level via an mRNA‐based therapy helps to overcome Gal‐Tox. To test this hypothesis in plants, we created Arabidopsis thaliana lines overexpressing USP from Pisum sativum. USP enzyme assays confirmed a threefold higher enzyme activity in the overexpression lines leading to a significant reduction of the Gal‐1P level in roots. Interestingly, the overexpression lines are phenotypically more sensitive to the exogenous addition of galactose (0.5 mmol L−1 Gal). Nucleotide sugar analysis via high‐performance liquid chromatography‐mass spectrometry revealed highly elevated UDP‐Gal levels in roots of seedlings grown on 1.5 mmol L−1 galactose versus 1.5 mmol L−1 sucrose. Analysis of plant cell wall glycans by comprehensive microarray polymer profiling showed a high abundance of antibody binding recognizing arabinogalactanproteins and extensins under Gal‐feeding conditions, indicating that glycoproteins are a major target for elevated UDP‐Gal levels in plants.

Untangling the impact of red wine maceration times on wine ageing. A multidisciplinary approach focusing on extended maceration in Shiraz wines

Phenolic composition of young red wines has been shown to play an important role in their ageing potential. Therefore, the modulation of phenolic extraction during maceration may influence the subsequent phenolic evolution of these wines. The present work aimed to evaluate the impact of three different maceration times on the phenolic levels and evolution observed over time, using spectrophotometric and chromatography methods, and the effect on the aroma, taste, and mouthfeel sensory properties using Projective Mapping. Additionally, grape cell wall deconstruction was monitored during the extended maceration phase by GC-MS and Comprehensive Comprehensive Microarray Polymer Profiling (CoMPP). Our findings demonstrated that longer maceration times did not always correspond to an increase in wine phenolic concentration, although the level of complexity of these molecules seemed to be higher. Additionally, continuous depectination and possible solubilisation of the pectin is observed during the extended maceration which may be influencing the sensory perception of these wines. Maceration time was also shown to influence the evolution of the polymeric fraction and sensory perception of the wines.

Tracking cell wall changes in wine and table grapes undergoing Botrytis cinerea infection using glycan microarrays.

The necrotrophic fungus Botrytis cinerea infects a broad range of fruit crops including domesticated grapevine Vitis vinifera cultivars. Damage caused by this pathogen is severely detrimental to the table and wine grape industries and results in substantial crop losses worldwide. The apoplast and cell wall interface is an important setting where many plant-pathogen interactions take place and where some defence-related messenger molecules are generated. Limited studies have investigated changes in grape cell wall composition upon infection with B. cinerea, with much being inferred from studies on other fruit crops. In this study, comprehensive microarray polymer profiling in combination with monosaccharide compositional analysis was applied for the first time to investigate cell wall compositional changes in the berries of wine (Sauvignon Blanc and Cabernet Sauvignon) and table (Dauphine and Barlinka) grape cultivars during Botrytis infection and tissue maceration. This was used in conjunction with scanning electron microscopy (SEM) and X-ray computed tomography (CT) to characterize infection progression. Grapes infected at veraison did not develop visible infection symptoms, whereas grapes inoculated at the post-veraison and ripe stages showed evidence of significant tissue degradation. The latter was characterized by a reduction in signals for pectin epitopes in the berry cell walls, implying the degradation of pectin polymers. The table grape cultivars showed more severe infection symptoms, and corresponding pectin depolymerization, compared with wine grape cultivars. In both grape types, hemicellulose layers were largely unaffected, as was the arabinogalactan protein content, whereas in moderate to severely infected table grape cultivars, evidence of extensin epitope deposition was present. Specific changes in the grape cell wall compositional profiles appear to correlate with fungal disease susceptibility. Cell wall factors important in influencing resistance may include pectin methylesterification profiles, as well as extensin reorganization.

The Influence of Hydrolytic Enzymes on Tannin Adsorption-Desorption onto Grape Cell Walls in a Wine-Like Matrix.

This study evaluates the capacity of four hydrolytic enzymes to limit the interactions between grape cell-walls and tannins and/or to favor tannin desorption. Adsorption and desorption tests were conducted by mixing a commercial seed tannin with purified skin cell-walls from Syrah grapes, in the presence or absence of hydrolytic enzymes, in a model-wine solution. The effects of the enzymes were evaluated by measuring the tannins in solution by High Performance Liquid Chromatography (HPLC) and the changes in the cell wall polysaccharide network by Comprehensive Microarray Polymer Profiling (COMPP) while the polysaccharides liberated from cell walls were analyzed by Size Exclusion Chromatography (SEC). The results showed that the enzymes limited the interaction between tannins and cell walls, especially cellulase, pectinase and xylanase, an effect associated with the cell wall structural modifications caused by the enzymes, which reduced their capacity to bind tannins. With regards to the tannin desorption process, enzymes did not play a significant role in liberating bound tannins. Those enzymes that showed the highest effect in limiting the adsorption of tannins and in disorganizing the cell wall structure, cellulase and pectinase, did not lead to a desorption of bound tannins, although they still showed a capacity of affecting cell wall structure. The results indicate that enzymes are not able to access those polysaccharides where tannins are bound, thus, they are not a useful tool for desorbing tannins from cell walls. The practical importance implications of these findings are discussed in the manuscript.

Overexpression of VviPGIP1 and NtCAD14 in Tobacco Screened Using Glycan Microarrays Reveals Cell Wall Reorganisation in the Absence of Fungal Infection.

The expression of Vitis vinifera polygalacturonase inhibiting protein 1 (VviPGIP1) in Nicotiana tabacum has been linked to modifications at the cell wall level. Previous investigations have shown an upregulation of the lignin biosynthesis pathway and reorganisation of arabinoxyloglucan composition. This suggests cell wall tightening occurs, which may be linked to defence priming responses. The present study used a screening approach to test four VviPGIP1 and four NtCAD14 overexpressing transgenic lines for cell wall alterations. Overexpressing the tobacco-derived cinnamyl alcohol dehydrogenase (NtCAD14) gene is known to increase lignin biosynthesis and deposition. These lines, particularly PGIP1 expressing plants, have been shown to lead to a decrease in susceptibility towards grey rot fungus Botrytis cinerea. In this study the aim was to investigate the cell wall modulations that occurred prior to infection, which should highlight potential priming phenomena and phenotypes. Leaf lignin composition and relative concentration of constituent monolignols were evaluated using pyrolysis gas chromatography. Significant concentrations of lignin were deposited in the stems but not the leaves of NtCAD14 overexpressing plants. Furthermore, no significant changes in monolignol composition were found between transgenic and wild type plants. The polysaccharide modifications were quantified using gas chromatography (GC–MS) of constituent monosaccharides. The major leaf polysaccharide and cell wall protein components were evaluated using comprehensive microarray polymer profiling (CoMPP). The most significant changes appeared at the polysaccharide and protein level. The pectin fraction of the transgenic lines had subtle variations in patterning for methylesterification epitopes for both VviPGIP1 and NtCAD14 transgenic lines versus wild type. Pectin esterification levels have been linked to pathogen defence in the past. The most marked changes occurred in glycoprotein abundance for both the VviPGIP1 and NtCAD14 lines. Epitopes for arabinogalactan proteins (AGPs) and extensins were notably altered in transgenic NtCAD14 tobacco.

Golgi-localized exo-β1,3-galactosidases involved in cell expansion and root growth in Arabidopsis

Plant arabinogalactan proteins (AGPs) are a diverse group of cell surface- and wall-associated glycoproteins. Functionally important AGP glycans are synthesized in the Golgi apparatus, but the relationships among their glycosylation levels, processing, and functionalities are poorly understood. Here, we report the identification and functional characterization of two Golgi-localized exo-β-1,3-galactosidases from the glycosyl hydrolase 43 (GH43) family in Arabidopsis thaliana GH43 loss-of-function mutants exhibited root cell expansion defects in sugar-containing growth media. This root phenotype was associated with an increase in the extent of AGP cell wall association, as demonstrated by Yariv phenylglycoside dye quantification and comprehensive microarray polymer profiling of sequentially extracted cell walls. Characterization of recombinant GH43 variants revealed that the exo-β-1,3-galactosidase activity of GH43 enzymes is hindered by β-1,6 branches on β-1,3-galactans. In line with this steric hindrance, the recombinant GH43 variants did not release galactose from cell wall-extracted glycoproteins or AGP-rich gum arabic. These results indicate that the lack of exo-β-1,3-galactosidase activity alters cell wall extensibility in roots, a phenotype that could be explained by the involvement of galactosidases in AGP glycan biosynthesis.

Non-GMO potato lines, synthesizing increased amylose and resistant starch, are mainly deficient in isoamylase debranching enzyme.

Summary Solanum tuberosum potato lines with high amylose content were generated by crossing with the wild potato species Solanum sandemanii followed by repeated backcrossing to Solanum tuberosum lines. The trait, termed increased amylose (IAm), was recessive and present after three generations of backcrossing into S. tuberosum lines (6.25% S. sandemanii genes). The tubers of these lines were small, elongated and irregular with small and misshaped starch granules and high sugar content. Additional backcrossing resulted in less irregular tuber morphology, increased starch content (4.3%–9.5%) and increased amylose content (29%–37.9%) but indifferent sugar content. The amylose in the IAm starch granules was mainly located in peripheral spots, and large cavities were found in the granules. Starch pasting was suppressed, and the digestion‐resistant starch (RS) content was increased. Comprehensive microarray polymer profiling (CoMPP) analysis revealed specific alterations of major pectic and glycoprotein cell wall components. This complex phenotype led us to search for candidate IAm genes exploiting its recessive trait. Hence, we sequenced genomic DNA of a pool of IAm lines, identified SNPs genome wide against the draft genome sequence of potato and searched for regions of decreased heterozygosity. Three regions, located on chromosomes 3, 7 and 10, respectively, displayed markedly less heterozygosity than average. The only credible starch metabolism‐related gene found in these regions encoded the isoamylase‐type debranching enzyme Stisa1. Decreased expression of mRNA (>500 fold) and reduced enzyme activity (virtually absent from IAm lines) supported Stisa1 as a candidate gene for IAm.

Analysis of Plant Cell Walls Using High-Throughput Profiling Techniques with Multivariate Methods.

Plant cell walls are composed of a number of coextensive polysaccharide-rich networks (i.e., pectin, hemicellulose, protein). Polysaccharide-rich cell walls are important in a number of biological processes including fruit ripening, plant-pathogen interactions (e.g., pathogenic fungi), fermentations (e.g., winemaking), and tissue differentiation (e.g., secondary cell walls). Applying appropriate methods is necessary to assess biological roles as for example in putative plant gene functional characterization (e.g., experimental evaluation of transgenic plants). Obtaining datasets is relatively easy, using for example gas chromatography-mass spectrometry (GC-MS) methods for monosaccharide composition, Fourier transform infrared spectroscopy (FT-IR) and comprehensive microarray polymer profiling (CoMPP); however, analyzing the data requires implementing statistical tools for large-scale datasets. We have validated and implemented a range of multivariate data analysis methods on datasets from tobacco, grapevine, and wine polysaccharide studies. Here we present the workflow from processing samples to acquiring data to performing data analysis (particularly principal component analysis (PCA) and orthogonal projection to latent structure (OPLS) methods).

The impact of carbohydrate-active enzymes on mediating cell wall polysaccharide-tannin interactions in a wine-like matrix.

Tannins are present in grape skins and seeds from where they are transferred into the must-wine matrix during the maceration stages of winemaking. However, tannin transfer is often incomplete. This could be due, among other reasons, to tannins becoming bound to grape cell wall polysaccharides, including soluble polymers, which are released during vinification and are present in high concentrations in the must/wine. The use of cell wall deconstructing enzymes offers the possibility of reducing these interactions, releasing more tannins into the final wine. The main aim of this study was to evaluate the optimal addition (individually, in combination or sequentially) of hydrolytic enzymes that would prevent tight polysaccharide-tannin associations. The use of comprehensive microarray polymer profiling (CoMPP) methodology provided key insights into how the enzyme treatments impacted the grape cell wall matrix and tannin binding. The results demonstrated that polygalacturonase + pectin-lyase promoted the highest release of tannins into solution.

Tracking polysaccharides during white winemaking using glycan microarrays reveals glycoprotein-rich sediments.

Winemaking results in a significant amount of sediments that are formed in the tanks, the vats and in the bottles before and after fermentation. Little is known about the biochemical composition of these sediments apart from the fact that they are assumed to be derived in large part from the grape matrix. Glycan microarray technology offers a relatively rapid means to track the polysaccharides from their origin in the grape material and throughout the various steps in the winemaking process. In this study Comprehensive Microarray Polymer Profiling (CoMPP) was used to investigate the glycan-rich composition of particularly white grapes during winemaking and then investigate the effects of recombinant and commercial enzyme formulations on wine sediment compositions. The gross lees or sediments produced in the absence of enzymes were found to be composed of an abundance of homogalacturonans, rhamnogalacturonans, arabinans and galactans in addition to an abundance of extensins and arabinogalactan proteins. The addition of enzymes was shown to strip off the homogalacturonan and much of the rhamnogalacturonan with its side chains revealing a sediment layer composed almost exclusively of extensins and arabinogalactan proteins. The effect of winemaking techniques was shown to have an effect on the glycan-rich wine sediment compositions and holds implications for the management of gross lees in a winery environment.

Dynamics of intracellular mannan and cell wall folding in the drought responses of succulent Aloe species.

Plants have evolved a multitude of adaptations to survive extreme conditions. Succulent plants have the capacity to tolerate periodically dry environments, due to their ability to retain water in a specialized tissue, termed hydrenchyma. Cell wall polysaccharides are important components of water storage in hydrenchyma cells. However, the role of the cell wall and its polysaccharide composition in relation to drought resistance of succulent plants are unknown. We investigate the drought response of leaf‐succulent Aloe (Asphodelaceae) species using a combination of histological microscopy, quantification of water content, and comprehensive microarray polymer profiling. We observed a previously unreported mode of polysaccharide and cell wall structural dynamics triggered by water shortage. Microscopical analysis of the hydrenchyma cell walls revealed highly regular folding patterns indicative of predetermined cell wall mechanics in the remobilization of stored water and the possible role of homogalacturonan in this process. The in situ distribution of mannans in distinct intracellular compartments during drought, for storage, and apparent upregulation of pectins, imparting flexibility to the cell wall, facilitate elaborate cell wall folding during drought stress. We conclude that cell wall polysaccharide composition plays an important role in water storage and drought response in Aloe.

Understanding Changes in Tomato Cell Walls in Roots and Fruits: The Contribution of Arbuscular Mycorrhizal Colonization.

Modifications in cell wall composition, which can be accompanied by changes in its structure, were already reported during plant interactions with other organisms, such as the mycorrhizal fungi. Arbuscular mycorrhizal (AM) fungi are among the most widespread soil organisms that colonize the roots of land plants, where they facilitate mineral nutrient uptake from the soil in exchange for plant-assimilated carbon. In AM symbiosis, the host plasma membrane invaginates and proliferates around all the developing intracellular fungal structures, and cell wall material is laid down between this membrane and the fungal cell surface. In addition, to improve host nutrition and tolerance/resistance to environmental stresses, AM symbiosis was shown to modulate fruit features. In this study, Comprehensive Microarray Polymer Profiling (CoMMP) technique was used to verify the impact of the AM symbiosis on the tomato cell wall composition both at local (root) and systemic level (fruit). Multivariate data analyses were performed on the obtained datasets looking for the effects of fertilization, inoculation with AM fungi, and the fruit ripening stage. Results allowed for the discernment of cell wall component modifications that were correlated with mycorrhizal colonization, showing a different tomato response to AM colonization and high fertilization, both at the root and the systemic level.

Deconstructing Wine Grape Cell Walls with Enzymes During Winemaking: New Insights from Glycan Microarray Technology.

Enzyme-aid maceration is carried out in most modern winemaking industries with a range of positive impacts on wine production. However, inconsistencies in enzyme efficiency are an issue complicated by unclear targets (limited information available on berry cell wall architecture of different cultivars) and the complex wine environment (i.e., fermenting must). Recent studies have been performed to develop a clearer picture of grape cell wall structures, maceration effects, and interactions between important wine compounds and grape-derived polysaccharides. This review highlights critically important recent studies on grape berry cell wall changes during ripening, the importance of enzymes during maceration (skin contact phase) and deconstruction processes that occur during alcoholic fermentation. The novelty of the Comprehensive Microarray Polymer Profiling (CoMPP) technique using cell wall probes (e.g., antibodies) as a method for following cell wall derived polymers during different biological and biotechnological processes is discussed. Recent studies, using CoMPP together with classical analytical methods, confirmed the developmental pattern of berry cell wall changes (at the polymer level) during grape ripening. This innovative technique were also used to track enzyme-assisted depectination of grape skins during wine fermentation and determine how this influence the release of wine favourable compounds. Furthermore, polysaccharides (e.g., arabinogalactan proteins) present in the final wine could be identified. Overall, CoMPP provides a much more enriched series of datasets compared to traditional approaches. Novel insights and future studies investigating grape cell wall and polyphenol interactions, and the tailoring of enzyme cocktails for consistent, effective and “customized” winemaking is advanced and discussed.

Nanofibers Produced from Agro-Industrial Plant Waste Using Entirely Enzymatic Pretreatments.

Cellulose fibers can be freed from the cell-wall skeleton via high-shear homogenization, to produce cellulose nanofibers (CNF) that can be used, for example, as the reinforcing phase in composite materials. Nanofiber production from agro-industrial byproducts normally involves harsh chemical-pretreatments and high temperatures to remove noncellulosic polysaccharides (20-70% of dry weight). However, this is expensive for large-scale processing and environmentally damaging. An enzyme-only pretreatment to obtain CNF from agro-industrial byproducts (potato and sugar beet) was developed with targeted commercial enzyme mixtures. It is hypothesized that cellulose can be isolated from the biomass, using enzymes only, due to the low lignin content, facilitating greater liberation of CNF via high-shear homogenization. Comprehensive Microarray Polymer Profiling (CoMPP) measured remaining extractable polysaccharides, showing that the enzyme-pretreatment was more successful at removing noncellulosic polysaccharides than alkaline- or acid-hydrolysis alone. While effective alone, the effect of the enzyme-pretreatment was bolstered via combination with a mild high-pH pretreatment. Dynamic rheology was used to estimate the proportion of CNF in resultant suspensions. Enzyme-pretreated suspensions showed 4-fold and 10-fold increases in the storage modulus for potato and sugar beet, respectively, compared to untreated samples. A greener yet facile method for producing CNF from vegetable waste is presented here.

Analyses of Aloe Polysaccharides Using Carbohydrate Microarray Profiling.

Background: As the popularity of Aloe vera extracts continues to rise, a desire to fully understand the individual polymer components of the leaf mesophyll, their relation to one another, and the effects they have on the human body are increasing. Polysaccharides present in the leaf mesophyll have been identified as the components responsible for the biological activities of A. vera, and they have been widely studied in the past decades. However, the commonly used methods do not provide the desired platform to conduct large comparative studies of polysaccharide compositions, as most of them require a complete or near-complete fractionation of the polymers. Objective: The objective for this study was to assess whether carbohydrate microarrays could be used for the high-throughput analysis of cell wall polysaccharides in aloe leaf mesophyll. Methods: The method we chose is known as comprehensive microarray polymer profiling (CoMPP) and combines the high-throughput capacity of microarray technology with the specificity of molecular probes. Results: Preliminary findings showed that CoMPP can successfully be used for high-throughput screening of aloe leaf mesophyll tissue. Seventeen species of Aloe and closely related genera were analyzed, and a clear difference in the polysaccharide compositions of the mesophyll tissues was seen. Conclusions: These preliminary data suggest that the polysaccharides vary between species and that true species of Aloe may differ from segregate genera.