Spatial relationships between polymers in Sitka spruce: proton spin-diffusion studies

The spatial arrangement of polymers in Sitka spruce (Picea sitchensis) was investigated by NMR proton spin-diffusion studies, supplemented by deuterium-exchange experiments monitored by FTIR spectroscopy. The FTIR spectra of earlywood sections after vapour-phase exchange with deuterium oxide showed that 43% of the hydroxyl groups were accessible to deuteration. This value is lower than predicted in the absence of aggregation of cellulose microfibrils into larger units, but greater than the predicted level of deuteration if 3.5-nm microfibrils surrounded by hemicellulose sheaths were aggregated into 4×4 arrays without space for deuterium oxide to penetrate between the microfibrils. The rate of proton spin diffusion between lignin and cellulose was consistent with the presence of microfibril arrays with approximately these dimensions and with lignin located outside them, in both earlywood and latewood. Proton spin-diffusion data for hemicelluloses were complicated by difficulties in assigning signals to glucomannans and xylans, but there was evidence for the spatial association of one group of hemicelluloses, including acetylated glucomannans, with cellulose surfaces, while another group of hemicelluloses was in spatial proximity to lignin. These data are consistent with a number of nanoscale models for the Sitka spruce cell wall, including a model in which glucomannans are associated with microfibril surfaces within the aggregate and water can penetrate partially between these surfaces, and one in which all non-cellulosic polymers and water are excluded from the interior of each microfibril aggregate

Structural studies of a fucogalactoxyloglucan from pinus radiata primary cell walls : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

1. The changes in carbohydrate composition of elongating Pinus radiata primary cell walls were investigated. In the hemicellulose B extracts, a large increase in the percentage of non-starch, non-cellulosic, glucose was found to occur on cessation of cell-wall elongation. 2. By fractionation of the hemicellulose B extracts, with a variety of methods involving precipitation from an aqueous solution, a xyloglucan was purified. This xyloglucan was the major hemicellulose of the Pinus radiata hypocotyl cell wall. 3. Characterisation studies on the xyloglucan involved: quantitative analysis of the monosaccharides derived by nitric acid/urea hydrolysis; identification of the partial hydrolysis products derived by trifluoroacetic acid hydrolysis; quantitation of the sugar linkages using methylation by the Hakomori method; and analysis of the anomeric configuration of component sugars using chromium trioxide oxidation. 4. From the results a tentative structure has been suggested for the xyloglucan, consisting of a backbone of B-D-gluco-pyranose residues linked together by 1-4 glycosidic bonds, and with sidechains of single xylose residues linked through C-6 of the glucose units. Galacto and fuco-1,2- galacto sidechains are attached to some of the xylose residues, probably through the C-2 of the xylose

Active Site Mapping of Xylan-Deconstructing Enzymes with Arabinoxylan Oligosaccharides Produced by Automated Glycan Assembly

Xylan-degrading enzymes are crucial for the deconstruction of hemicellulosic biomass, making the hydrolysis products available for various industrial applications such as the production of biofuel. To determine the substrate specificities of these enzymes, we prepared a collection of complex xylan oligosaccharides by automated glycan assembly. Seven differentially protected building blocks provided the basis for the modular assembly of 2-substituted, 3-substituted, and 2-/3-substituted arabino- and glucuronoxylan oligosaccharides. Elongation of the xylan backbone relied on iterative additions of C4-fluorenylmethoxylcarbonyl (Fmoc) protected xylose building blocks to a linker-functionalized resin. Arabinofuranose and glucuronic acid residues have been selectively attached to the backbone using fully orthogonal 2-(methyl)naphthyl (Nap) and 2-(azidomethyl)benzoyl (Azmb) protecting groups at the C2 and C3 hydroxyls of the xylose building blocks. The arabinoxylan oligosaccharides are excellent tools to map the active site of glycosyl hydrolases involved in xylan deconstruction. The substrate specificities of several xylanases and arabinofuranosidases were determined by analyzing the digestion products after incubation of the oligosaccharides with glycosyl hydrolases.Fil: Senf, Deborah. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; AlemaniaFil: Ruprecht, Colin. Max Planck Institut für Kolloid und Grenzflächenforschung; AlemaniaFil: de Kruijff, Goswinus H. M.. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; Alemania. University Mainz. Institute of Institute of Organic Chemistry, Johannes Gutenberg; AlemaniaFil: Simonetti, Sebastián Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina. Max Planck Institut für Kolloid und Grenzflächenforschung; AlemaniaFil: Schuhmacher, Frank. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; AlemaniaFil: Seeberger, Peter H.. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; AlemaniaFil: Pfrengle, Fabian. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; Alemani

Extraction and structural study of hemicellulose B from tall fescue (Festuca arundinacea) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at Massey University

1.1 Hemicellulose The structural carbohydrates are mixtures of polysaccharides which together with lignin, constitute the cell wall. Usually they are divided into three fractions: pectic substances, hemicellulose and cellulose.1 Pectins, widely distributed in land plants are characterised by a main chain of 1 – 4 linked galacturonic acid units. They may also contain rhamnose, galactose, xylose, arabinose and fucose in varying amounts. These sugars form part the main chain in some pectins and branch chains in others.2 The pectic triad consists of galacturonan, araban and galactan. Pectic substances occur without exception in all higher plants. It is possible that pectic substances may serve as protective agents for natural rubber particles and may be important food reserve for the plant.3 They are found most abundantly in the primary cell walls and in the intercellular layer.4 [From Introduction

Fungi isolated from Miscanthus and sugarcane: biomass conversion, fungal enzymes, and hydrolysis of plant cell wall polymers.

BackgroundBiofuel use is one of many means of addressing global change caused by anthropogenic release of fossil fuel carbon dioxide into Earth's atmosphere. To make a meaningful reduction in fossil fuel use, bioethanol must be produced from the entire plant rather than only its starch or sugars. Enzymes produced by fungi constitute a significant percentage of the cost of bioethanol production from non-starch (i.e., lignocellulosic) components of energy crops and agricultural residues. We, and others, have reasoned that fungi that naturally deconstruct plant walls may provide the best enzymes for bioconversion of energy crops.ResultsPreviously, we have reported on the isolation of 106 fungi from decaying leaves of Miscanthus and sugarcane (Appl Environ Microbiol 77:5490-504, 2011). Here, we thoroughly analyze 30 of these fungi including those most often found on decaying leaves and stems of these plants, as well as four fungi chosen because they are well-studied for their plant cell wall deconstructing enzymes, for wood decay, or for genetic regulation of plant cell wall deconstruction. We extend our analysis to assess not only their ability over an 8-week period to bioconvert Miscanthus cell walls but also their ability to secrete total protein, to secrete enzymes with the activities of xylanases, exocellulases, endocellulases, and beta-glucosidases, and to remove specific parts of Miscanthus cell walls, that is, glucan, xylan, arabinan, and lignin.ConclusionThis study of fungi that bioconvert energy crops is significant because 30 fungi were studied, because the fungi were isolated from decaying energy grasses, because enzyme activity and removal of plant cell wall components were recorded in addition to biomass conversion, and because the study period was 2 months. Each of these factors make our study the most thorough to date, and we discovered fungi that are significantly superior on all counts to the most widely used, industrial bioconversion fungus, Trichoderma reesei. Many of the best fungi that we found are in taxonomic groups that have not been exploited for industrial bioconversion and the cultures are available from the Centraalbureau voor Schimmelcultures in Utrecht, Netherlands, for all to use

Polysaccharide utilization loci and nutritional specialization in a dominant group of butyrate-producing human colonic Firmicutes

Acknowledgements The Rowett Institute of Nutrition and Health (University of Aberdeen) receives financial support from the Scottish Government Rural and Environmental Sciences and Analytical Services (RESAS). POS is a PhD student supported by the Scottish Government (RESAS) and the Science Foundation Ireland, through a centre award to the APC Microbiome Institute, Cork, Ireland. Data Summary The high-quality draft genomes generated in this work were deposited at the European Nucleotide Archive under the following accession numbers: 1. Eubacterium rectale T1-815; CVRQ01000001–CVRQ0100 0090: http://www.ebi.ac.uk/ena/data/view/PRJEB9320 2. Roseburia faecis M72/1; CVRR01000001–CVRR010001 01: http://www.ebi.ac.uk/ena/data/view/PRJEB9321 3. Roseburia inulinivorans L1-83; CVRS01000001–CVRS0 100 0151: http://www.ebi.ac.uk/ena/data/view/PRJEB9322Peer reviewedPublisher PD

Glycan complexity dictates microbial resource allocation in the large intestine.

The structure of the human gut microbiota is controlled primarily through the degradation of complex dietary carbohydrates, but the extent to which carbohydrate breakdown products are shared between members of the microbiota is unclear. We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan. Characterization of the extensive xylan degrading apparatus expressed by B. ovatus reveals that the breakdown of the polysaccharide by the human gut microbiota is significantly more complex than previous models suggested, which were based on the deconstruction of xylans containing limited monosaccharide side chains. Our report presents a highly complex and dynamic xylan degrading apparatus that is fine-tuned to recognize the different forms of the polysaccharide presented to the human gut microbiota.This work was supported in part by grants to D.N.B. (BBSRC BB/G016186/1) and H.J.G. (Wellcome Trust WT097907AIA).This is the final version. It was first published by NPG at http://dx.doi.org/10.1038/ncomms848

A novel xylan degrading β-D-xylosidase: purification and biochemical characterization

Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular b-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. b-xylosidase is a glycoprotein (39 % carbohydrate content) and has a molecular mass of 137 kDa by SDS-PAGE, with optimal temperature and pH at 70 C and 3.0–5.5, respectively.b-xylosidase was stable in acidic pH (3.0–6.0) and 70 C for 1 h. The enzyme was activated by 5 mM MnCl2 (28 %)and MgCl2 (20 %) salts. The b-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-b- D-xylopyranoside, exhibiting apparent Km and Vmax values of 0.66 mM and 39 U (mg protein)-1 respectively, and to a lesser extent p-nitrophenyl-b-D-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources,suggesting a novel b-D-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by b-xylosidase. TLC confirmed the capacity.This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and the Conselho de Desenvolvimento Científico e Tecnológico (CNPq). J. A. J. and M. L. T. M. P are Research Fellows of CNPq. M. M. was a recipient of a FAPESP fellowship and this work is part of her Doctoral Thesis. It is also part of the project SISBIOTA CNPq: 563260/2010-6 and FAPESP: 2010/52322-3