Galactan (Potato)

Commercially-supplied potato galactan (PG) is widely used as a model polysaccharide in various bioactivity studies. However, results using this galactan are not always consistent with the stated composition. Here, we assessed its composition by fractionating this commercial PG and purified its primary components: PG-A, PG-B and PG-Cp with weight-averaged molecular weights of 430, 93, and 11.3 kDa, respectively. PG-Cp consists of free β-1,4-galactan chains, whereas PG-A and PG-B are type I rhamnogalacturonans with long β-1,4-galactan side chains of up to 80 Gal residues and short β-1,4-galactan side chains of 0 to 3 Gal residues that display a “trees in lawn” pattern. Structures of these polysaccharides correlate well with their activities in terms of galectin-3 binding and gut bacterial growth assays. Our study clarifies the confusion related to commercial PG, with purified fractions serving as better model polysaccharides in bioactivity investigations.

The deposition of proteins in the form of insoluble amyloid fibril aggregates is linked to a range of diseases. The supramolecular architecture of such deposits is governed by the propagation of β-strands in the direction of protofilament growth. In the present study, we analyze the structural changes of hen egg-white lysozyme fibrils upon their interactions with a range of polysaccharides, using AFM and FTIR spectroscopy. Linear anionic polysaccharides, such as κ-carrageenan and sodium alginate, are shown to be capable to disaggregate protofilaments with eventual protein renaturation. The results help to understand the mechanism of amyloid disaggregation and create a platform for both the development of new therapeutic agents for amyloidose treatment, and the design of novel functional protein–polysaccharide complex-based nanomaterials.

The immune system produces a diverse collection of antiglycan antibodies that are critical for host defense. At present, however, we know very little about the binding properties, origins, and sequences of these antibodies because of a lack of access to a variety of defined individual antibodies. To address this challenge, we used a glycan microarray with over 800 different components to screen a panel of 516 human monoclonal antibodies that had been randomly cloned from different B-cell subsets originating from healthy human subjects. We obtained 26 antiglycan antibodies, most of which bound microbial carbohydrates. The majority of the antiglycan antibodies identified in the screen displayed selective binding for specific glycan motifs on our array and lacked polyreactivity. We found that antiglycan antibodies were about twice as likely than expected to originate from IgG⁺ memory B cells, whereas none were isolated from naïve, early emigrant, or immature B cells. Therefore, our results indicate that certain B-cell subsets in our panel are enriched in antiglycan antibodies, and IgG⁺ memory B cells may be a promising source of such antibodies. Furthermore, some of the newly identified antibodies bound glycans for which there are no reported monoclonal antibodies available, and these may be useful as research tools, diagnostics, or therapeutic agents. Overall, the results provide insight into the types and properties of antiglycan antibodies produced by the human immune system and a framework for the identification of novel antiglycan antibodies in the future.

Background: Shipworms are marine xylophagus bivalve molluscs, which can live on a diet solely of wood due to their ability to produce plant cell wall-degrading enzymes. Bacterial carbohydrate-active enzymes (CAZymes), synthesised by endosymbionts living in specialised shipworm cells called bacteriocytes and located in the animal’s gills, play an important role in wood digestion in shipworms. However, the main site of lignocellulose digestion within these wood-boring molluscs, which contains both endogenous lignocellulolytic enzymes and prokaryotic enzymes, is the caecum, and the mechanism by which bacterial enzymes reach the distant caecum lumen has remained so far mysterious. Here, we provide a characterisation of the path through which bacterial CAZymes produced in the gills of the shipworm Lyrodus pedicellatus reach the distant caecum to contribute to the digestion of wood. Results: Through a combination of transcriptomics, proteomics, X-ray microtomography, electron microscopy studies and in vitro biochemical characterisation, we show that wood-digesting enzymes produced by symbiotic bacteria are localised not only in the gills, but also in the lumen of the food groove, a stream of mucus secreted by gill cells that carries food particles trapped by filter feeding to the mouth. Bacterial CAZymes are also present in the crystalline style and in the caecum of their shipworm host, suggesting a unique pathway by which enzymes involved in a symbiotic interaction are transported to their site of action. Finally, we characterise in vitro four new bacterial glycosyl hydrolases and a lytic polysaccharide monooxygenase identified in our transcriptomic and proteomic analyses as some of the major bacterial enzymes involved in this unusual biological system. Conclusions: Based on our data, we propose that bacteria and their enzymes are transported from the gills along the food groove to the shipworm’s mouth and digestive tract, where they aid in wood digestion.

Lactobacillus plantarum is a useful microorganism that metabolizes galactose-containing polysaccharides. Genome analysis has shown that L. plantarum contains four β-galactosidase-related genes. Here, we cloned the β-galactosidase gene that encodes the glycoside hydrolase family 2 (GH2) enzyme LpGal2. Recombinant LpGal2 (rLpGal2, 72 kDa) is a homodimer with maximal enzymatic activity at pH 7.0 and 50°C. Under these conditions, rLpGal2 hydrolyzes p-nitrophenyl-β-D-galactopyranoside (pNPβGal) with a specific activity of 2.16 × 10⁻³ U/mg and substrate specificity for β-1,6-galactobiose to produce D-Galactose. In addition, rLpGal2 can also hydrolyze β-1,6-galactan to D-Galactose, whereas other galactose-containing oligosaccharides and polysaccharides tested could not be hydrolyzed. This finding demonstrates that LpGal2 functions as an exo-β-1,6-galactosidase with narrow substrate specificity. To our knowledge, this is the first report of a β-galactosidase derived from L. plantarum with exo-β-1,6-galactosidase activity that has potential application for structure analysis of polysaccharides.

Comparison of Salvia miltiorrhiza polysaccharides (SMPs) from different geographical origins in China (Henan, Hebei, Shandong, Sichuan, Shaanxi) was performed using high performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector (HPSEC-MALLS-RID), saccharide mapping based on polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) and combined with principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Based on the results of HPSEC-MALLS/RI, the relative content of SMPs showed a significant difference between different geographical origins, however, the molecular weight of SMPs showed almost no significance. SMPs can be discriminated as five regions after PACE coupled with OPLS-DA models analysis of endo-1,5-α-arabinanase hydrolysates. Moreover, all the PACE fingerprint indicated that 1,4-β-D-Galp, 1,5-α-Araf, 1,4-α-D-GalAp and 1,4-β-D-Glcp linkages existed in SMPs.

Microorganisms residing within a biofilm become more tolerant to antibiotics and other types of adverse impact, and biofilm formation by pathogenic bacteria is an important problem of current medicine. Polysaccharides that prevent biofilm formation are among the promising candidates to help tackle this problem. Earlier we demonstrated the ability of a potato polysaccharide galactan to inhibit biofilm formation by a Pseudomonas aeruginosa clinical isolate. Here we investigate the effect of potato galactan on P. aeruginosa biofilms in more detail. Microscopic analysis indicated that the galactan did not interfere with the adhesion of bacterial cells to the substrate but prevented the build-up of bacterial biomass. Moreover, the galactan not only inhibited biofilm formation, but partially destroyed pre-formed biofilms. Presumably, this activity of the galactan was due to the excessive aggregation of bacterial cells, which prohibited the formation and maintenance of proper biofilm architecture, or due to some other mechanisms of biofilm structure remodeling. This led to an unexpected effect, i.e., P. aeruginosa biofilms treated with an antibiotic and the galactan retained more viable bacterial cells compared to biofilms treated with the antibiotic alone. Galactan is the first polysaccharide demonstrated to exert such effect on bacterial biofilms.

Galectin-3 (Gal-3) can induce T-cell activation and apoptosis and plays a role in tumor immune tolerance. Here, we demonstrate that ginseng pectins selectively inhibit Gal-3-induced T-cell apoptosis, while not affecting T-cell activation. This finding stands in contrast to that from the use of modified citrus pectin (MCP) and potato galactan (P-galactan) that inhibit both. Whereas PKC/ERK and ROS/ERK pathways are involved in both T-cell activation and apoptosis, the Ras/PI3K/Akt pathway is unique to T-cell activation. Ginseng pectins selectively inhibit the ROS/ERK pathway. Using the Sarcomar-180 mouse model in which Gal-3 expression is increased, we found that ginseng pectins (but not MCP or P-galactan) significantly promote T-cell proliferation and IL-2 expression, and inhibit tumor growth by 45%. These in vivo data correlate well with selective effects of pectins on Gal-3-mediated T-cell apoptosis and activation. Our study suggests a novel approach for the development of polysaccharide-based agents that target Gal-3 function.

Background: Cellulose and hemicellulose are the two largest components in lignocellulosic biomass. Enzymes with activities towards cellulose and xylan have attracted great interest in the bioconversion of lignocellulosic biomass, since they have potential in improving the hydrolytic performance and reducing the enzyme costs. Exploring glycoside hydrolases (GHs) with good thermostability and activities on xylan and cellulose would be beneficial to the industrial production of biofuels and bio-based chemicals. Results: A novel GH10 enzyme (XynA) identified from a xylanolytic strain Bacillus sp. KW1 was cloned and expressed. Its optimal pH and temperature were determined to be pH 6.0 and 65°C. Stability analyses revealed that XynA was stable over a broad pH range (pH 6.0-11.0) after being incubated at 25°C for 24 h. Moreover, XynA retained over 95% activity after heat treatment at 60°C for 60 h, and its half-lives at 65°C and 70°C were about 12 h and 1.5 h, respectively. More importantly, in terms of substrate specificity, XynA exhibits hydrolytic activities towards xylans, microcrystalline cellulose (filter paper and Avicel), carboxymethyl cellulose (CMC), cellobiose, p-nitrophenyl-β-D-cellobioside (pNPC), and p-nitrophenyl-β-D-glucopyranoside (pNPG). Furthermore, the addition of XynA into commercial cellulase in the hydrolysis of pretreated corn stover resulted in remarkable increases (the relative increases may up to 90%) in the release of reducing sugars. Finally, it is worth mentioning that XynA only shows high amino acid sequence identity (88%) with rXynAHJ14, a GH10 xylanase with no activity on CMC. The similarities with other characterized GH10 enzymes, including xylanases and bifunctional xylanase/cellulase enzymes, are no more than 30%. Conclusions: XynA is a novel thermostable GH10 xylanase with a wide substrate spectrum. It displays good stability in a broad range of pH and high temperatures, and exhibits activities towards xylans and a wide variety of cellulosic substrates, which are not found in other GH10 enzymes. The enzyme also has high capacity in saccharification of pretreated corn stover. These characteristics make XynA a good candidate not only for assisting cellulase in lignocellulosic biomass hydrolysis, but also for the research on structure-function relationship of bifunctional xylanase/cellulase.

Type II arabinogalactan (AG) is a soluble prebiotic fiber stimulating the proliferation of bifidobacteria in the human gut. Larch AG, which is comprised of type II AG, is known to be utilized as an energy source for Bifidobacterium longum subsp. longum (B. longum). We have previously characterized GH43_24 exo-β-1,3-galactanase (Bl1,3Gal) for the degradation of type II AG main chains in B. longum JCM1217. In this study, we characterized GH30_5 exo-β-1,6-galactobiohydrolase (Bl1,6Gal) and GH43_22 α-L-arabinofuranosidase (BlArafA), which are degradative enzymes for type II AG side chains in cooperation with exo-β-1,3-galactanase. The recombinant exo-β-1,6-galactobiohydrolase specifically released β-1,6-galactobiose (β-1,6-Gal₂) from the nonreducing terminal of β-1,6-galactooligosaccharides, and the recombinant α-L-arabinofuranosidase released arabinofuranose (Araf) from α-1,3-Araf -substituted β-1,6-galactooligosaccharides. β-1,6-Gal₂ was additively released from larch AG by the combined use of type II AG degradative enzymes, including Bl1,3Gal, Bl1,6Gal, and BlArafA. The gene cluster encoding the type II AG degradative enzymes is conserved in all B. longum strains, but not in other bifidobacterial species. The degradative enzymes for type II AG side chains are thought to be important for the acquisition of type II AG in B. longum.

The interactions between cellulose and pectin polysaccharides in primary plant cell walls are not fully understood, although several recent studies indicate that they might play an important role in wall properties. Studying polysaccharide interactions in planta is challenging, due to the complexity and heterogeneity of plant materials. Therefore, to investigate these interactions and the implications for the rheological properties of cell walls, we have taken a bottom-up approach in which cellulose/pectin composites are created either by adsorption of pectin polysaccharides (arabinan, galactan, homogalacturonan DE 69, homogalacturonan DE 33 and pectin DE 33) on cellulose-coated sensors in a quartz crystal microbalance with dissipation monitoring (QCM-D) or by incorporation of pectin during in vivo cellulose synthesis by Komagataeibacter bacteria. The viscoelastic behavior of the adsorbed layers was analyzed by applying the Voigt model to the QCM-D data, whilst the bulk viscoelastic properties of bacterial cellulose/pectin composites were studied by small amplitude oscillatory shear rheology. Our results show that all of the pectin polysaccharides studied have the ability to adsorb on the cellulose surfaces. The viscoelastic properties of the adsorbed layer varied depending on the substitution and degree of esterification of the pectin polysaccharides. Additionally, oscillatory rheology results showed that all bacterial cellulose-pectin composites had a gel nature (G′ > G″) with moduli varying in line with QCM-D determined viscoelasticity. Our interpretation of the results provides a better understanding of pectin-cellulose interactions and has implications for primary plant cell wall material properties.

Rhamnogalacturonan I (RG-I), a polysaccharide found in different types of plant cell walls, fulfills specific functions, the structural basis of which remains unclear. Generalized 2D correlation FTIR spectroscopy with dehydration was employed to reveal the structure and interactions in flax RG-I solution and microwave treated gel. Varying water content allowed emphasizing a role of solvent in maintaining different structures. In the gel, 2D correlation maps prove the existence of a conformationally uniform highly hydrated structure. Such a structure is supposed to correspond to non-associated galactan helices stabilized by rare junctions. In colloidal solution the side chains of RG-I associate heterogeneously due to constrains imposed by stiff backbone. Galactan-enriched fraction of RG-I with enzymatically cleaved backbone revealed the tendency of galactan chains to strongly associate in solution. The obtained results shed light on the possible role of backbone and side chains in RG-I spatial organization and confirm the sensitivity and potential of 2D correlation FTIR spectroscopy to probe local ordered structures in non-crystalline polysaccharides.

Prebiotics are selectively fermented by the gastrointestinal microflora, resulting in benefits to human health. The seed mucilage of Hyptis suaveolens contains neutral and acidic polysaccharides in a ratio of 1:1. The neutral polysaccharides consist of galactose, glucose and mannose whereas the acidic polysaccharides contain fucose, xylose and 4-O-methylglucuronic acid -residues. The growth of probiotics in the presence of total, acidic or neutral polysaccharides and oligosaccharides was tested using turbidity measurements. The majority (11 out of 14) of the tested probiotic strains significantly grew in the neutral fraction. Growth occurred with some time delay, but may be longer lasting than with other lower molecular prebiotics. The extent of growth increased with neutral polysaccharides from H. suaveolens corresponding to the externally available galactose units (20%). In conclusion, neutral poly- and oligosaccharides from H. suaveolens have a prebiotic potential characterized by a delayed but long lasting effect.

Background: The Aspergillus niger genome contains a large repertoire of genes encoding carbohydrate active enzymes (CAZymes) that are targeted to plant polysaccharide degradation enabling A. niger to grow on a wide range of plant biomass substrates. Which genes need to be activated in certain environmental conditions depends on the composition of the available substrate. Previous studies have demonstrated the involvement of a number of transcriptional regulators in plant biomass degradation and have identified sets of target genes for each regulator. In this study, a broad transcriptional analysis was performed of the A. niger genes encoding (putative) plant polysaccharide degrading enzymes. Microarray data focusing on the initial response of A. niger to the presence of plant biomass related carbon sources were analyzed of a wild-type strain N402 that was grown on a large range of carbon sources and of the regulatory mutant strains δxlnR, δaraR, δamyR, δrhaR and δgalX that were grown on their specific inducing compounds. Results: The cluster analysis of the expression data revealed several groups of co-regulated genes, which goes beyond the traditionally described co-regulated gene sets. Additional putative target genes of the selected regulators were identified, based on their expression profile. Notably, in several cases the expression profile puts questions on the function assignment of uncharacterized genes that was based on homology searches, highlighting the need for more extensive biochemical studies into the substrate specificity of enzymes encoded by these non-characterized genes. The data also revealed sets of genes that were upregulated in the regulatory mutants, suggesting interaction between the regulatory systems and a therefore even more complex overall regulatory network than has been reported so far. Conclusions: Expression profiling on a large number of substrates provides better insight in the complex regulatory systems that drive the conversion of plant biomass by fungi. In addition, the data provides additional evidence in favor of and against the similarity-based functions assigned to uncharacterized genes.

Galactan-rich rhamnogalacturonan I (RG I), exhibiting promising health benefits, is the most abundant polysaccharide in potato pulp by-product. In the present study, the microwave-assisted alkaline extraction of galactan-rich RG I was investigated. Solid/liquid ratio was identified as the most significant parameter affecting linearly yield and galactose/rhamnose contents. Microwave power and solid/liquid ratio exhibited a significant adverse interactive effect on the yield. Galactose content of extracted polysaccharides can be modulated by compromising between KOH concentration and extraction time, which exhibited adverse interaction. Optimum conditions were identified using the established predicted models and consisted of treatment of potato cell wall at solid/liquid ratio of 2.9% (w/v) with 1.5 M KOH under microwave power of 36.0 W for 2.0 min. Yield of intact galactan-rich RG I of 21.6% and productivity of 192.0 g/L h were achieved. The functional properties of RG I-rich polysaccharides were comparable or superior to potato galactan and oranges homogalacturonan.

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.

The production of glycated lysozyme (LZM), with galactose, galactooligosaccharides (GOSs) and potato galactan through the Maillard reaction, was investigated. The percent blocked lysine, estimated from the furosine content, reached a maximum value of 11.2% for LZM:galactan conjugates after 1 day incubation at a a_w of 0.65. A maximum percent blocked lysine of 7.0 and 13.5% were obtained for LZM:galactose/GOS conjugates at a lower a_w of 0.45 after 3 and 7 days, respectively. However, the low percent blocked lysine and the high protein aggregation index of LZM:galactose/GOS conjugates at a_w 0.79 and 0.65 revealed the prevalence of the degradation of the Amadori compounds and the protein cross-linking. Mass spectrometry of LZM conjugates revealed the formation of different glycoforms. Glycated LZMs containing up to seven galactose moieties were formed; while only mono- and diglycated LZMs with GOSs were detected. 2–3 mol of galactan were conjugated to 1 mol of LZM. Response surface methodology, based on a 5-level and 3-factor central composite design, revealed that molar ratio and temperature were the most significant variables for the glycation of LZM with GOSs. The optimal conditions leading to a high percent blocked lysine (16.11%) with a low protein aggregation index (0.11) were identified: temperature of 49.5°C, LZM:GOS molar ratio of 1:9 and a_w of 0.65. To the best of our knowledge, this is the first study on the optimization of LZM glycation with GOSs.

Potato pulp is a high-volume side-stream from industrial potato starch manufacturing. Enzymatically solubilized β-1,4-galactan-rich potato pulp polysaccharides of molecular weights >100 kDa (SPPP) are highly bifidogenic in human fecal sample fermentations in vitro. The objective of the present study was to use potato β-1,4-galactan and the SPPP as substrates for enzymatic production of potentially prebiotic compounds of lower and narrower molecular weight. A novel endo-1,4-β-galactanase from Emericella nidulans (anamorph Aspergillus nidulans), GH family 53, was produced in a recombinant Pichia pastoris strain. The enzyme was purified by Cu²⁺ affinity chromatography and its optimal reaction conditions were determined to pH 5 and 49°C via a statistical experimental design. The specific activity of the E. nidulans enzyme expressed in P. pastoris was similar to that of an endo-1,4-β-galactanase from Aspergillus niger used as benchmark. The E. nidulans enzyme expressed in P. pastoris generated a spectrum poly- and oligo-saccharides which were fractionated by membrane filtration. The potential growth promoting properties of each fraction were evaluated by growth of beneficial gut microbes and pathogenic bacteria. All the galactan- and SPPP-derived products promoted the growth of probiotic strains of Bifidobacterium longum and Lactobacillus acidophilus and generally did not support the propagation of Clostridium perfringens in single culture fermentations. Notably the growth of B. longum was significantly higher (p<0.05) or at least as good on galactan- and SPPP-derived products as fructooligosaccharides (FOS). Except in one case these products did not support the growth of the pathogen Cl. perfringens to any significant extent.

The extracellular bga1-encoded β-galactosidase of Hypocrea jecorina (Trichoderma reesei) was overexpressed under the pyruvat kinase (pki1) promoter region and purified to apparent homogeneity. The monomeric enzyme is a glycoprotein with a molecular mass of 118.8 ± 0.5 kDa (MALDI-MS) and an isoelectric point of 6.6. Bga1 is active with several disaccharides, e.g. lactose, lactulose and galactobiose, as well as with aryl- and alkyl-β-D-galactosides. Based on the catalytic efficiencies, lactitol and lactobionic acid are the poorest substrates and o-nitrophenyl-β-D-galactoside and lactulose are the best. The pH optimum for the hydrolysis of galactosides is 5.0, and the optimum temperature was found to be 60°C. Bga1 is also capable of releasing D-galactose from β-galactans and is thus actually a galacto-β-D-galactanase. β-Galactosidase is inhibited by its reaction product D-galactose and the enzyme also shows a significant transferase activity which results in the formation of galacto-oligosaccharides.

The structure of arabinan and galactan domains in association with cellulose microfibrils was investigated using enzymatic and alkali degradation procedures. Sugar beet and potato cell wall residues (called ‘natural’ composites), rich in pectic neutral sugar side chains and cellulose, as well as ‘artificial’ composites, created by in vitro adsorption of arabinan and galactan side chains onto primary cell wall cellulose, were studied. These composites were sequentially treated with enzymes specific for pectic side chains and hot alkali. The degradation approach used showed that most of the arabinan and galactan side chains are in strong interaction with cellulose and are not hydrolysed by pectic side chain-degrading enzymes. It seems unlikely that isolated arabinan and galactan chains are able to tether adjacent microfibrils. However, cellulose microfibrils may be tethered by different pectic side chains belonging to the same pectic macromolecule.