A fibrolytic potential in the human ileum mucosal microbiota revealed by functional metagenomics.
Patrascu, O., Béguet-Crespel, F., Marinelli, L., Le Chatelier, E., Abraham, A., Leclerc, M., Klopp, C., Terrapon, N., Henrissat, B., Blottière, H. M., Doré, J. & Christel Béra-Maillet. (2017). Scientific Reports, 7, 40248.
The digestion of dietary fibers is a major function of the human intestinal microbiota. So far this function has been attributed to the microorganisms inhabiting the colon, and many studies have focused on this distal part of the gastrointestinal tract using easily accessible fecal material. However, microbial fermentations, supported by the presence of short-chain fatty acids, are suspected to occur in the upper small intestine, particularly in the ileum. Using a fosmid library from the human ileal mucosa, we screened 20,000 clones for their activities against carboxymethylcellulose and xylans chosen as models of the major plant cell wall (PCW) polysaccharides from dietary fibres. Eleven positive clones revealed a broad range of CAZyme encoding genes from Bacteroides and Clostridiales species, as well as Polysaccharide Utilization Loci (PULs). The functional glycoside hydrolase genes were identified, and oligosaccharide break-down products examined from different polysaccharides including mixed-linkage β-glucans. CAZymes and PULs were also examined for their prevalence in human gut microbiome. Several clusters of genes of low prevalence in fecal microbiome suggested they belong to unidentified strains rather specifically established upstream the colon, in the ileum. Thus, the ileal mucosa-associated microbiota encompasses the enzymatic potential for PCW polysaccharide degradation in the small intestine.
RT-CaCCO process: an improved CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature.
Shiroma, R., Park, J. Y., Al-Haq, M. I., Arakane, M., Ike, M. & Tokuyasu, K. (2011). Bioresource Technology, 102(3), 2943-2949.
We improved the CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature (RT). We firstly optimized the RT-lime pretreatment for the lignocellulosic part. When the ratio of lime/dry-biomass was 0.2 (w/w), the RT lime-pretreatment for 7-d resulted in an effect on the enzymatic saccharification of cellulose and xylan equivalent to that of the pretreatment at 120°C for 1 h. Sucrose, starch and β-1,3-1,4-glucan, which could be often detected in rice straw, were mostly stable under the RT-lime pretreatment condition. Then, the pretreatment condition in the conventional CaCCO process was modified by the adaptation of the optimized RT lime-pretreatment, resulting in significantly better carbohydrate recoveries via enzymatic saccharification than those of the CaCCO process (120°C for 1 h). Thus, the improved CaCCO process (the RT-CaCCO process) could preserve/pretreat the feedstock at RT in a wet form with minimum loss of carbohydrates.
Real-time imaging of cellulose reorientation during cell wall expansion in Arabidopsis roots.
Anderson, C. T., Carroll, A., Akhmetova, L. & Somerville, C. (2010). Plant Physiology, 152(2), 787-796.
Cellulose forms the major load-bearing network of the plant cell wall, which simultaneously protects the cell and directs its growth. Although the process of cellulose synthesis has been observed, little is known about the behavior of cellulose in the wall after synthesis. Using Pontamine Fast Scarlet 4B, a dye that fluoresces preferentially in the presence of cellulose and has excitation and emission wavelengths suitable for confocal microscopy, we imaged the architecture and dynamics of cellulose in the cell walls of expanding root cells. We found that cellulose exists in Arabidopsis (Arabidopsis thaliana) cell walls in large fibrillar bundles that vary in orientation. During anisotropic wall expansion in wild-type plants, we observed that these cellulose bundles rotate in a transverse to longitudinal direction. We also found that cellulose organization is significantly altered in mutants lacking either a cellulose synthase subunit or two xyloglucan xylosyltransferase isoforms. Our results support a model in which cellulose is deposited transversely to accommodate longitudinal cell expansion and reoriented during expansion to generate a cell wall that is fortified against strain from any direction.
Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12.
Gloster, T. M., Ibatullin, F. M., Macauley, K., Eklöf, J. M., Roberts, S., Turkenburg, J. P., Bjørnvad, M. E., Jørgensen, P. L., Danielsen, S., Johansen, K. S., Borchert, T. V., Wilson, K. S., Brumer, H. & Davies, G. J. (2007). Journal of Biological Chemistry, 282(26), 19177-19189.
The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed “hemicellulose.” One such hemicellulose is xyloglucan, which displays a β-1,4-linked D-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand-free and xyloglucan-oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (β/α)8 and β-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the β-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzyme is similarly competent in the degradation of unbranched glucans. In the case of the (β/ α)8 GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.
Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module.
Gunnarsson, L. C., Montanier, C., Tunnicliffe, R. B., Williamson, M. P., Gilbert, H. J., Nordberg, K. E. & Ohlin, M. (2007). Biochem. J, 406(2), 209-214.
Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.
Recombinant expression and enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides.
Master, E. R., Rudsander, U. J., Zhou, W., Henriksson, H., Divne, C., Denman, S., Wilson D. B. & Teeri, T. T. (2004). Biochemistry, 43(31), 10080-10089.
PttCel9A is a membrane-bound, family 9 glycosyl hydrolase from Populus tremula x tremuloides that is upregulated during secondary cell wall synthesis. The catalytic domain of PttCel9A, Δ1-105PttCel9A, was purified, and its activity was compared to TfCel9A and TfCel9B from Thermobifida fusca. Since aromatic amino acids involved in substrate binding at subsites −4, −3, and −2 are missing in PttCel9A, the activity of TfCel9A mutant enzymes W256S, W209A, and W313G was also investigated. Δ1-105PttCel9A hydrolyzed a comparatively narrow range of polymeric substrates, and the preferred substrate was (carboxymethyl)cellulose 4M. Moreover, Δ1-105PttCel9A did not hydrolyze oligosaccharides shorter than cellopentaose, whereas TfCel9A and TfCel9B hydrolyzed cellotetraose and cellotriose, respectively. These data suggest that the preferred substrates of PttCel9A are long, low-substituted, soluble cellulosic polymers. At 30°C and pH 6.0, the kcat for cellohexaose of Δ1-105PttCel9A, TfCel9A, and TfCel9B were 0.023 ± 0.001, 16.9 ± 2.0, and 1.3 ± 0.2, respectively. The catalytic efficiency (kcat/km) of TfCel9B was 39% of that of TfCel9A, whereas the catalytic efficiency of Δ1-105PttCel9A was 0.04% of that of TfCel9A. Removing tryptophan residues at subsites −4, −3, and −2 decreased the efficiency of cellohexaose hydrolysis by TfCel9A. Mutation of W313 to G had the most drastic effect, producing a mutant enzyme with 1% of the catalytic efficiency of TfCel9A. The apparent narrow substrate range and catalytic efficiency of PttCel9A are correlated with a lack of aromatic amino acids in the substrate binding cleft and may be necessary to prevent excessive hydrolysis of cell wall polysaccharides during cell wall formation.
Neoglycolipid-Based “Designer” Oligosaccharide Microarrays to Define β-Glucan Ligands for Dectin-1.
Palma, A. S., Zhang, Y., Childs, R. A., Campanero-Rhodes, M. A., Liu, Y., Feizi, T. & Chai, W. (2012). Carbohydrate Microarrays, 808(2), 337-359.
In this chapter, we describe the key steps of the “designer” oligosaccharide microarray approach we followed to prove the carbohydrate binding activity and define the oligosaccharide ligands for Dectin-1, an atypical C-type lectin-like signaling receptor of the mammalian innate immune system with a key role in anti-fungal immunity. The term “designer” microarray, which we introduced in the course of the Dectin-1 study refers to a microarray of oligosaccharide probes generated from ligand-bearing glycoconjugates to reveal the oligosaccharide ligands they harbor, so that these can be isolated and characterized. Oligosaccharide probes were generated from two polysaccharides, one that was bound by Dectin-1 and known to be rich in β1,3-glucose sequence and another that was not bound and was rich in β1,6-glucose sequence and served as a negative control. The approach involved: classic ELISA-type binding assays to select the polysaccharides; partial depolymerization of the polysaccharides by chemical hydrolysis; fractionation by size of the glucan oligosaccharides obtained and determination of their chain lengths by mass spectrometry; detection of Dectin-1 ligand-positive and ligand-negative oligosaccharides using the neoglycolipid (NGL) technology; methylation analysis of oligosaccharides to derive glucose linkage information, and incorporation of the newly generated glucan oligosaccharide probes into microarrays encompassing diverse mammalian-type and exogenous sequences for microarray analysis of Dectin-1.
Cloning of an endoglycanase gene from Paenibacillus cookii and characterization of the recombinant enzyme.
Shinoda, S., Kanamasa, S. & Arai, M. (2012). Biotechnology Letters, 34(2), 281-286.
An endoglycanase gene of Paenibacillus cookii SS-24 was cloned and sequenced. This Pgl8A gene had an open reading frame of 1,230 bp that encoded a putative signal sequence (31 amino acids) and mature enzyme (378 amino acids: 41,835 Da). The enzyme was most homologous to a β-1,3-1,4-glucanase of Bacillus circulans WL-12 with 84% identity. The recombinant enzyme hydrolyzed carboxymethyl cellulose, swollen celluloses, chitosan and lichenan but not Avicel, chitin powder or xylan. With chitosan as the substrate, the optimum temperature and hydrolysis products of the recombinant enzyme varied at pH 4.0 and 8.0. This is the first report that characterizes chitosanase activity under different pH conditions.
Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: biological implications for cell wall metabolism.
Baumann, M. J., Eklöf, J. M., Michel, G., Kallas, Å. M., Teeri, T. T., Czjzek, M. & Brumer, H. (2007). The Plant Cell, 19(6), 1947-1963.
High-resolution, three-dimensional structures of the archetypal glycoside hydrolase family 16 (GH16) endo-xyloglucanases Tm-NXG1 and Tm-NXG2 from nasturtium (Tropaeolum majus) have been solved by x-ray crystallography. Key structural features that modulate the relative rates of substrate hydrolysis to transglycosylation in the GH16 xyloglucan-active enzymes were identified by structure–function studies of the recombinantly expressed enzymes in comparison with data for the strict xyloglucan endo-transglycosylase Ptt-XET16-34 from hybrid aspen (Populus tremula × Populus tremuloides). Production of the loop deletion variant Tm-NXG1-ΔYNIIG yielded an enzyme that was structurally similar to Ptt-XET16-34 and had a greatly increased transglycosylation:hydrolysis ratio. Comprehensive bioinformatic analyses of XTH gene products, together with detailed kinetic data, strongly suggest that xyloglucanase activity has evolved as a gain of function in an ancestral GH16 XET to meet specific biological requirements during seed germination, fruit ripening, and rapid wall expansion.
A comparative study on structure–function relations of mixed-linkage (1→ 3),(1→ 4) linear β-D-glucans.
Lazaridou, A., Biliaderis, C. G., Micha-Screttas, M. & Steele, B. R. (2004). Food Hydrocolloids, 18(5), 837-855.
The effects of fine structure and molecular size on the rheological properties of six mixed-linkage (1→3), (1→4)-β-D-glucans (β-glucans) in the solution and gel state were studied. Molecular size characterization was carried out with high-performance size exclusion chromatography combined with a refractive index detector. Samples were divided into two groups according to the values of apparent molecular weight (Mw) of the peak fraction of the main eluting peak calculated as 200×103 for an oat, a barley, and a wheat β-glucan and ∼100×103 for an oat and a barley β-glucan, and a lichenan sample. All polysaccharides analyzed by 2D NMR spectroscopy and high-performance anion-exchange chromatography of the cellulosic oligomers released by the action of lichenase showed the typical fine structure of mixed-linkage linear (1→3), (1→4)-β-D-glucan. Following lichenase digestion of β-glucans, the molar ratios of tri- to tetrasaccharides (DP3/DP4) were found to follow the order of lichenan (24.5)>wheat (3.7)>barley (2.8–3.0)>oat (2.1). Differences in critical concentration (c* *), viscosity, viscoelastic and shear thinning properties among samples were dependent mainly on differences in molecular size of the polymeric chains as well as on the β-glucan fine structure. All β-glucan isolates were able to form gels, as probed by dynamic rheometry; with decreasing molecular size and increasing DP3/DP4 ratio, the gelation time decreased and the gelation rate (IE=[d log G'/dt ]max) increased. Differential scanning calorimetry (DSC) showed that cereal β-glucan gels exhibit rather broad endothermic gel→sol transitions at 55–80°C, while lichenan gels give a sharper transition, implying a more cooperative process. The DSC kinetic data showed similar responses to that from dynamic rheometry; the rate of development of the endotherm increased with increasing DP3/DP4 ratio of the polysaccharide. Furthermore, the storage modulus (G′) and the apparent melting enthalpy values (plateau ΔH) increased with decreasing molecular size and with increasing DP3/DP4 ratio. The melting temperature of the gel network, as determined by DSC and dynamic rheometry, was found to increase with the molecular size and the DP3/DP4 ratio of β-glucans; the Tm for lichenan was ∼89°C and for cereal β-glucans varied in the narrow range of ∼65–72°C. Large deformation mechanical tests (compression mode) up to failure revealed an increase in strength and a decrease in brittleness of mixed-linkage β-glucan gels with increasing DP3/DP4 ratio and molecular size of the polysaccharide.
Evaluation of structure in the formation of gels by structurally diverse (1→3)(1→4)-β-D-glucans from four cereal and one lichen species.
Tosh, S. M., Brummer, Y., Wood, P. J., Wang, Q. & Weisz, J. (2004). Carbohydrate Polymers, 57(3), 249-259.
The (1→3)(1→4)-β-D-glucans from four cereal sources (oats, wheat, barley and rye) and one lichen source (Icelandic moss) were used to test two proposed structurally based hypotheses about the gelling mechanism of these polymers. Structures were evaluated using high performance anion exchange chromatography of the oligosaccharide fragments released by a (1→3)(1→4)-β-D-glucan-4-glucanohydrolase. This determined the relative amounts of cellodextrin units, of different degrees of polymerisation, which are joined by β-(1→3) linkages in the intact polysaccharide chain. Oat β-glucan had the lowest β-(1→3)-linked cellotriosyl unit content and lichenan had the highest. Strong correlations were found between the fraction of β-(1→3)-linked cellotriosyl units in the β-glucans and the elasticity of 6% gels in water, as measured by dynamic rheometry. Differential scanning calorimetry showed that the β-(1→3)-linked cellotriosyl unit content was also correlated with the onset and peak temperatures when 6% β-glucan gels were melted. No correlation was found between the longer (DP 6–9) β-(1→3)-linked cellodextrin oligosaccharide content and either the gel elasticity or melting characteristics. These findings are consistent with a model in which runs of consecutive β-(1→3)-linked cellotriosyl units form the junction zones in the gel network, but not with a model in which longer β-(1→3)-linked cellodextrins associate, as in cellulose fibres, to produce the gel network. Microscopic images of the β-glucan gels from the five species revealed that the microstructure was not homogeneous in any of the samples, which may be related to the variability in the enthalpy of melting of gels. There was a coarsening of gel structure as the β-(1→3)-linked cellotriosyl unit content increased.