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  • 151.
    Kallas, Åsa M.
    et al.
    KTH, School of Biotechnology (BIO).
    Baumann, Martin J.
    KTH, School of Biotechnology (BIO).
    Fäldt, Jenny
    KTH.
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO).
    Denman, Stuart
    KTH.
    Mellerowicz, Ewa J.
    Nishikubo, Nobuyushi
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO).
    Enzymatic characterization of a recombinant xyloglucan endotransglycosylase PttXET16-35 from Populus tremula x tremuloidesManuscript (Other academic)
  • 152.
    Kanoth, Bipinbal Parambath
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Claudino, Mauro
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Johansson, Mats
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Berglund, Lars A.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Biocomposites from Natural Rubber: Synergistic Effects of Functionalized Cellulose Nanocrystals as Both Reinforcing and Cross-Linking Agents via Free-Radical Thiol-ene Chemistry2015In: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 7, no 30, p. 16303-16310Article in journal (Refereed)
    Abstract [en]

    Natural rubber/cellulose nanocrystals (NR/CNCs) form true biocomposites from renewable resources and are demonstrated to show significantly improved thermo-mechanical properties and reduced stress-softening. The nanocomposites were prepared from chemically functionalized CNCs bearing thiols. CNCs served as both reinforcing and cross-linking agents in the NR matrix, and the study was designed to prove the cross-linking function of modified CNCs. CNCs were prepared from cotton, and the cross-linkable mercapto-groups were introduced onto the surface of CNCs by esterification. Nanocomposite films were prepared by dispersing the modified CNCs (m-CNCs) in NR matrix by solution casting. The cross-links at the filler matrix (m-CNCs NR) interface were generated by photochemically initiated thiol-ene reactions as monitored by real-time FTIR analysis. The synergistic effects of reinforcement and chemical cross-linking at the m-CNCs NR interface on structure, thermo-mechanical, and stress-softening behavior were investigated. Methods included field emission scanning electron microscopy (FE-SEM), swelling tests, dynamic mechanical analysis, and tensile tests. Compared to biocomposites from NR with unmodified CNCs, the NR/m-CNCs nanocomposites showed 2.4-fold increase in tensile strength, 1.6-fold increase in strain-to-failure, and 2.9-fold increase in work-of-fracture at 10 wt % of m-CNCs in NR.

  • 153.
    Kishani, Saina
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Xu, Wenyang
    Abo Akad Univ, Lab Wood & Paper Chem, Johan Gadolin Proc Chem Ctr, FI-20500 Turku, Finland..
    Xu, Chunlin
    Abo Akad Univ, Lab Wood & Paper Chem, Johan Gadolin Proc Chem Ctr, FI-20500 Turku, Finland..
    Wågberg, Lars
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Solubility of Softwood Hemicelluloses2018In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 19, no 4, p. 1245-1255Article in journal (Refereed)
    Abstract [en]

    It is demonstrated that the molecular solubility of softwood hemicelluloses is significantly influenced by pretreatment of the fibers, extraction, and downstream processing. To quantify these effects, four hemicellulose samples were extracted from different thermomechanical pulps of Norway spruce. The molecular solubility of the samples was characterized by size and molar mass distributions, and the morphology of the molecules was studied using high resolution microscopy techniques. All extracted samples were well dispersed in aqueous media creating transparent dispersions, but dynamic light scattering measurements showed that molecular solubility can only be achieved using specific pretreatments and extractions. The procedure yields acetylated galactoglucomannan (AcGGM)-rich hemicelluloses with an average molar mass of 21-35 kDa and a diameter up to 10 nm but also shows that water is a poor solvent for this sample since an association is detected as soon as the concentration is about 20 g/L. These associated hemicellulose dispersions are still absolutely dear on visual inspection, underlining the need for careful measurement when assessing the solubility of wood hemicelluloses.

  • 154. Kisonen, Victor
    et al.
    Xu, Chunlin
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Eklund, Patrik
    Lindqvist, Hanna
    Sundberg, Anna
    Pranovich, Andrey
    Sinkkonen, Jari
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Willför, Stefan
    Cationised O-acetyl galactoglucomannans: Synthesis and characterisation2014In: Carbohydrate Polymers, ISSN 0144-8617, E-ISSN 1879-1344, Vol. 99, p. 755-764Article in journal (Refereed)
    Abstract [en]

    Water-soluble O-acetyl-galactoglucomannans (GGMs) can be obtained from Norway spruce by hot-water-extraction of the wood or as a side product by ultrafiltration of mechanical pulping waters. Cationic and amphiphilic polysaccharides and their derivatives are of interest for a number of applications and thus quaternary nitrogen moieties with cationic charge were grafted onto GGMs in the heterogeneous reaction to render a cationic polyelectrolyte. The degree of substitution was measured by elemental analysis of nitrogen, by quantitative C-13 NMR and interestingly also by polyelectrolyte titration and the results were congruent. NMR, matrix-assisted laser desorption/ionisation mass spectroscopy (MALDI-TOF-MS), and FT-IR analysis were used to characterise the product. THF or DMSO with water enhanced the reaction efficiency and decreased M-w reduction in comparison to plain water as a reaction media. Cationised GGM was also successfully acetylated. The cationic derivatives of hemicelluloses can potentially be utilised as polyelectrolyte layers in packaging and pharmaceutical applications.

  • 155. Kittl, Roman
    et al.
    Sygmund, Christoph
    Halada, Petr
    Volc, Jindrich
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Haltrich, Dietmar
    Peterbauer, Clemens K.
    Molecular cloning of three pyranose dehydrogenase-encoding genes from Agaricus meleagris and analysis of their expression by real-time RT-PCR2008In: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983, Vol. 53, no 2, p. 117-127Article in journal (Refereed)
    Abstract [en]

    Sugar oxidoreductases such as cellobiose dehydrogenase or pyranose oxidase are widespread enzymes among fungi, whose biological function is largely speculative. We investigated a similar gene family in the mushroom Agaricus meleagris and its expression under various conditions. Three genes (named pdh1, pdh2 and pdh3) putatively encoding pyranose dehydrogenases were isolated. All three genes displayed a conserved structure and organization, and the respective cDNAs contained ORFs translating into polypeptides of 602 or 600 amino acids. The N-terminal sections of all three genes encode putative signal peptides consistent with the enzymes extracellular secretion. We cultivated the fungus on different carbon sources and analyzed the mRNA levels of all three genes over a period of several weeks using real-time RT-PCR. The glyceraldehyde-3-phosphate dehydrogenase gene from A. meleagris was also isolated and served as reference gene. pdh2 and pdh3 are essentially transcribed constitutively, whereas pdh1 expression is upregulated upon exhaustion of the carbon source; pdh1 appears to be additionally regulated under conditions of oxygen limitation. These data are consistent with an assumed role in lignocellulose degradation.

  • 156.
    Kochumalayil, Joby
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Engineering Sciences (SCI), Centres, VinnExcellence Center BiMaC Innovation.
    Sehaqui, Houssine
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Berglund, Lars A.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Tamarind seed xyloglucan: a thermostable high-performance biopolymer from non-food feedstock2010In: Journal of Materials Chemistry, ISSN 0959-9428, E-ISSN 1364-5501, Vol. 20, no 21, p. 4321-4327Article in journal (Refereed)
    Abstract [en]

    Polysaccharide biopolymers from renewable resources are of great interest as replacements for petroleum-based polymers since they have lower cradle-to-grave non-renewable energy use and greenhouse gas emissions. Starch is widely used as a packaging material but is based on food resources such as potato or corn, and suffers from high sensitivity to water vapor even under ambient conditions. For the first time, xyloglucan (XG) from tamarind seed waste is explored as an alternative high-performance biopolymer from non-food feedstock. XG is purified, and dissolved in water to cast films. Moisture sorption isotherms, tensile tests and dynamic mechanical thermal analysis are performed. Glycerol plasticization toughening and enzymatic modification (partial removal of galactose in side chains of XG) are attempted as means of modification. XG films show much lower moisture sorption than the amylose component in starches. Stiffness and strength are very high, with considerable ductility and toughness. The thermal stability is exceptionally high and is approaching 250 degrees C. Glycerol plasticization is effective already at 10% glycerol. These observations point towards the potential of XG as a "new'' biopolymer from renewable non-food plant resources for replacement of petroleum-based polymers.

  • 157.
    Kuang, Guanglin
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tu, Yaoquan
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Computational studies of the binding profile of phosphoinositide PtdIns (3,4,5) P-3 with the pleckstrin homology domain of an oomycete cellulose synthase2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 20555Article in journal (Refereed)
    Abstract [en]

    Saprolegnia monoica is a model organism to investigate Saprolegnia parasitica, an important oomycete which causes considerable loss in aquaculture every year. S. monoica contains cellulose synthases vital for oomycete growth. However, the molecular mechanism of the cellulose biosynthesis process in the oomycete growth is still poorly understood. Some cellulose synthases of S. monoica, such as SmCesA2, are found to contain a plecsktrin homology (PH) domain, which is a protein module widely found in nature and known to bind to phosphoinositides, a class of signaling compounds involved in many biological processes. Understanding the molecular interactions between the PH domain and phosphoinositides would help to unravel the cellulose biosynthesis process of oomycetes. In this work, the binding profile of PtdIns (3,4,5) P-3, a typical phosphoinositide, with SmCesA2-PH was studied by molecular docking, molecular dynamics and metadynamics simulations. PtdIns (3,4,5) P-3 is found to bind at a specific site located at beta 1, beta 2 and beta 1-beta 2 loop of SmCesA2-PH. The high affinity of PtdIns (3,4,5) P-3 to SmCesA2-PH is contributed by the free phosphate groups, which have electrostatic and hydrogenbond interactions with Lys88, Lys100 and Arg102 in the binding site.

  • 158.
    Kuang, Guanglin
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Liang, Lijun
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology. Zhejiang University, China.
    Brown, Christian
    KTH, School of Biotechnology (BIO), Glycoscience.
    Wang, Qi
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, Australia.
    Tu, Yaoquan
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Insight into the adsorption profiles of the Saprolegnia monoica chitin synthase MIT domain on POPA and POPC membranes by molecular dynamics simulation studies2016In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 18, no 7, p. 5281-5290Article in journal (Refereed)
    Abstract [en]

    The critical role of chitin synthases in oomycete hyphal tip growth has been established. A microtubule interacting and trafficking (MIT) domain was discovered in the chitin synthases of the oomycete model organism, Saprolegnia monoica. MIT domains have been identified in diverse proteins and may play a role in intracellular trafficking. The structure of the Saprolegnia monoica chitin synthase 1 (SmChs1) MIT domain has been recently determined by our group. However, although our in vitro assay identified increased strength in interactions between the MIT domain and phosphatidic acid (PA) relative to other phospholipids including phosphatidylcholine (PC), the mechanism used by the MIT domain remains unknown. In this work, the adsorption behavior of the SmChs1 MIT domain on POPA and POPC membranes was systematically investigated by molecular dynamics simulations. Our results indicate that the MIT domain can adsorb onto the tested membranes in varying orientations. Interestingly, due to the specific interactions between MIT residues and lipid molecules, the binding affinity to the POPA membrane is much higher than that to the POPC membrane. A binding hotspot, which is critical for the adsorption of the MIT domain onto the POPA membrane, was also identified. The lower binding affinity to the POPC membrane can be attributed to the self-saturated membrane surface, which is unfavorable for hydrogen-bond and electrostatic interactions. The present study provides insight into the adsorption profile of SmChs1 and additionally has the potential to improve our understanding of other proteins containing MIT domains.

  • 159.
    Kuang, Guanglin
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Liang, Lijun
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Brown, Christian
    KTH, School of Biotechnology (BIO), Glycoscience.
    Wang, Qi
    Tu, Yaoquan
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tu, Yaoquan
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Insight into the adsorption profiles of the Saprolegnia practica chitin synthase MIT domain on POPA and POPC membranes by molecular dynamics simulation studiesManuscript (preprint) (Other academic)
  • 160. Kujawa, Magdalena
    et al.
    Ebner, Heidemarie
    Leitner, Christian
    Hallberg, B. Martin
    Prongjit, Methinee
    Sucharitakul, Jeerus
    Ludwig, Roland
    Rudsander, Ulla
    Peterbauer, Clemens
    Chaiyen, Pimchai
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 46, p. 35104-35115Article in journal (Refereed)
    Abstract [en]

    Pyranose2-oxidase(P2Ox) participates in fungal lignin degradation by producing the H2O2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose- and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regios-electivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower k(cat), and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose. Based on the details of 2-fluoro-2-deoxy-D-glucose binding in position for oxidation at C3, we also outline a probable binding mode for D-glucose positioned for regioselective oxidation at C2. The tentative determinant for discriminating between the two binding modes is the position of the O6 hydroxyl group, which in the C2-oxidation mode can make favorable interactions with Asp(452) in the substrate loop and, possibly, a nearby arginine residue (Arg(472)). We also substantiate our hypothesis with steady-state kinetics data for the alanine replacements of Asp(452) and Arg(472) as well as the double alanine 452/472 mutant.

  • 161. Kujawa, Magdalena
    et al.
    Volc, Jindrich
    Halada, Petr
    Sedmera, Petr
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Sygmund, Christoph
    Leitner, Christian
    Peterbauer, Clemens
    Haltrich, Dietmar
    Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma2007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 3, p. 879-894Article in journal (Refereed)
    Abstract [en]

    We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K-m). The enzyme may play a role in lignocellulose degradation.

  • 162.
    Kumar, Sanjiv
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. Department of Medicine, UConn Health, Farmington, CT, United States.
    Caimano, M. J.
    Anand, A.
    Dey, A.
    Hawley, K. L.
    Ledoyt, M. E.
    La Vake, C. J.
    Cruz, A. R.
    Ramirez, L. G.
    Paštěková, L.
    Bezsonova, I.
    Šmajs, D.
    Salazar, J. C.
    Radolf, J. D.
    Sequence variation of rare outer membrane protein β-barrel domains in clinical strains provides insights into the evolution of treponema pallidum subsp. Pallidum, the syphilis spirochete: QC 201811212018In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, no 3, article id e01006-18Article in journal (Refereed)
    Abstract [en]

    In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum, the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC, tprD, and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 (tp0548) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum. They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development. IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum, little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops.

  • 163.
    Larsbrink, Johan
    KTH, School of Biotechnology (BIO), Glycoscience.
    Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The focus of this thesis is a comparative study of approaches in discovery of carbohydrate-active enzymes (CAZymes). CAZymes synthesise, bind to, and degrade all the multitude of carbohydrates found in nature. As such, when aiming for sustainable methods to degrade plant biomass for the generation of biofuels, for which there is a strong drive in society, CAZymes are a natural source of environmentally friendly molecular tools.

    In nature, microorganisms are the principal degraders of carbohydrates. Not only do they degrade plant matter in forests and aquatic habitats, but also break down the majority of carbohydrates ingested by animals. These symbiotic microorganisms, known as the microbiota, reside in animal digestive tracts in immense quantities, where one of the key nutrient sources is complex carbohydrates. Thus, microorganisms are a plentiful source of CAZymes, and strategies in the discovery of new enzymes from bacterial sources have been the basis for the work presented here, combined with biochemical characterisation of several enzymes.

    Novel enzymatic activities for the glycoside hydrolase family 31 have been described as a result of the initial projects of the thesis. These later evolved into projects studying bacterial multi-gene systems for the partial or complete degradation of the heterogeneous plant polysaccharide xyloglucan. These systems contain, in addition to various hydrolytic CAZymes, necessary binding-, transport-, and regulatory proteins. The results presented here show, in detail, how very complex carbohydrates can efficiently be degraded by bacterial enzymes of industrial relevance.

  • 164.
    Larsbrink, Johan
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Screening and activity profiling of the glycoside hydrolase family 31 members from Bacteroides thetaiotaomicron reveal novel specificitiesManuscript (preprint) (Other academic)
  • 165.
    Larsbrink, Johan
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Izumi, Atsushi
    Hemsworth, Glyn R.
    Davies, Gideon J.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals alpha-Transglucosylase Activity in Glycoside Hydrolase Family 312012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 52, p. 43288-43299Article in journal (Refereed)
    Abstract [en]

    The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these alpha-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in alpha-glucan side chain (de) branching, regulation of oligo-and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-alpha-glucan 4-alpha-glucosyltransferase from GH31. Distinct from 1,4-alpha-glucan 6-alpha-glucosyltransferases (EC 2.4.1.24) and 4-alpha-glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from alpha(1 -> 4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-beta-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme.

  • 166.
    Larsbrink, Johan
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Izumi, Atsushi
    Ibatullin, Farid M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Nakhai, Azadeh
    KTH, School of Biotechnology (BIO), Glycoscience.
    Gilbert, Harry J.
    Davies, Gideon J.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Structural and enzymatic characterization of a glycoside hydrolase family 31 alpha-xylosidase from Cellvibrio japonicus involved in xyloglucan saccharification2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 436, p. 567-580Article in journal (Refereed)
    Abstract [en]

    The desire for improved methods of biomass conversion into fuels and feedstocks has re-awakened interest in the enzymology of plant cell wall degradation. The complex polysaccharide xyloglucan is abundant in plant matter, where it may account for up to 20% of the total primary cell wall carbohydrates. Despite this, few studies have focused on xyloglucan saccharification, which requires a consortium of enzymes including endo-xyloglucanases, alpha-xylosidases, beta-galactosidases and alpha-L-fucosidases, among others. In the present paper, we show the characterization of Xy131A, a key alpha-xylosidase in xyloglucan utilization by the model Gram-negative soil saprophyte Cellvibrio japonicus. CjXy131A exhibits high regiospecificity for the hydrolysis of XGOs (xylogluco-oligosaccharides), with a particular preference for longer substrates. Crystallographic structures of both the apo enzyme and the trapped covalent 5-fluoro-beta-xylosyl-enzyme intermediate, together with docking studies with the XXXG heptasaccharide, revealed, for the first time in GH31 (glycoside hydrolase family 31), the importance of PA14 domain insert in the recognition of longer oligosaccharides by extension of the active-site pocket. The observation that CjXy131A was localized to the outer membrane provided support for a biological model of xyloglucan utilization by C. japonicas, in which XGOs generated by the action of a secreted endo-xyloglucanase are ultimately degraded in close proximity to the cell surface. Moreover, the present study diversifies the toolbox of glycosidases for the specific modification and saccharification of cell wall polymers for biotechnological applications.

  • 167.
    Larsbrink, Johan
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Lundqvist, Magnus
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Identification and characterization of a locus in Cellvibrio japonicus involved in xylogluco-oligosaccharide saccharificationManuscript (preprint) (Other academic)
  • 168.
    Larsbrink, Johan
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Rogers, Theresa E.
    Hemsworth, Glyn R.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Tauzin, Alexandra S.
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Klinter, Stefan
    KTH, School of Biotechnology (BIO), Glycoscience.
    Pudlo, Nicholas A.
    Urs, Karthik
    Koropatkin, Nicole M.
    Creagh, A. Louise
    Haynes, Charles A.
    Kelly, Amelia G.
    Nilsson Cederholm, Stefan
    KTH, School of Biotechnology (BIO), Glycoscience.
    Davies, Gideon J.
    Martens, Eric C.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes2014In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 506, no 7489, p. 498-502Article in journal (Refereed)
    Abstract [en]

    A well-balanced human diet includes a significant intake of non-starch polysaccharides, collectively termed 'dietary fibre', from the cell walls of diverse fruits and vegetables(1). Owing to the paucity of alimentary enzymes encoded by the human genome(2), our ability to derive energy from dietary fibre depends on the saccharification and fermentation of complex carbohydrates by the massive microbial community residing in our distal gut(3,4). The xyloglucans (XyGs) are a ubiquitous family of highly branched plant cell wall polysaccharides(5,6) whose mechanism(s) of degradation in the human gut and consequent importance in nutrition have been unclear(1,7,8). Here we demonstrate that a single, complex gene locus in Bacteroides ovatus confers XyG catabolism in this common colonic symbiont. Through targeted gene disruption, biochemical analysis of all predicted glycoside hydrolases and carbohydrate-binding proteins, and three-dimensional structural determination of the vanguard endo-xyloglucanase, we reveal the molecular mechanisms through which XyGs are hydrolysed to component monosaccharides for further metabolism. We also observe that orthologous XyG utilization loci (XyGULs) serve as genetic markers of XyG catabolism in Bacteroidetes, that XyGULs are restricted to a limited number of phylogenetically diverse strains, and that XyGULs are ubiquitous in surveyed human metagenomes. Our findings reveal that the metabolism of even highly abundant components of dietary fibre may be mediated by niche species, which has immediate fundamental and practical implications for gut symbiont population ecology in the context of human diet, nutrition and health(9-12).

  • 169.
    Larsbrink, Johan
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience.
    McKee, Laurens S.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Klinter, Stefan
    KTH, School of Biotechnology (BIO), Glycoscience.
    Nilsson Cederholm, Stefan
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    et al.,
    A discrete genetic locus confers select Bacteriodetes with a niche role in xyloglucan metabolism in the human gutManuscript (preprint) (Other academic)
  • 170.
    Larsbrink, Johan
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Thompson, Andrew J.
    Lundqvist, Magnus
    KTH, School of Biotechnology (BIO), Glycoscience.
    Gardner, Jeffrey G.
    Davies, Gideon J.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. Univ British Columbia, Canada; .
    A complex gene locus enables xyloglucan utilization in the model saprophyte Cellvibrio japonicus2014In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 94, no 2, p. 418-433Article in journal (Refereed)
    Abstract [en]

    The degradation of plant biomass by saprophytes is an ecologically important part of the global carbon cycle, which has also inspired a vast diversity of industrial enzyme applications. The xyloglucans (XyGs) constitute a family of ubiquitous and abundant plant cell wall polysaccharides, yet the enzymology of XyG saccharification is poorly studied. Here, we present the identification and molecular characterization of a complex genetic locus that is required for xyloglucan utilization by the model saprophyte Cellvibrio japonicus. In harness, transcriptomics, reverse genetics, enzyme kinetics, and structural biology indicate that the encoded cohort of an -xylosidase, a -galactosidase, and an -l-fucosidase is specifically adapted for efficient, concerted saccharification of dicot (fucogalacto)xyloglucan oligosaccharides following import into the periplasm via an associated TonB-dependent receptor. The data support a biological model of xyloglucan degradation by C. japonicus with striking similarities - and notable differences - to the complex polysaccharide utilization loci of the Bacteroidetes.

  • 171.
    Larsbrink, Johan
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Tuveng, T. R.
    Pope, P. B.
    Bulone, V.
    Eijsink, V. G. H.
    Brumer, Harry
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Proteomic insights into mannan degradation and protein secretion by the forest floor bacterium Chitinophaga pinensis2017In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 156, p. 63-74Article in journal (Refereed)
    Abstract [en]

    Together with fungi, saprophytic bacteria are central to the decomposition and recycling of biomass in forest environments. The Bacteroidetes phylum is abundant in diverse habitats, and several species have been shown to be able to deconstruct a wide variety of complex carbohydrates. The genus Chitinophaga is often enriched in hotspots of plant and microbial biomass degradation. We present a proteomic assessment of the ability of Chitinophaga pinensis to grow on and degrade mannan polysaccharides, using an agarose plate-based method of protein collection to minimise contamination with exopolysaccharides and proteins from lysed cells, and to reflect the realistic setting of growth on a solid surface. We show that select Polysaccharide Utilisation Loci (PULs) are expressed in different growth conditions, and identify enzymes that may be involved in mannan degradation. By comparing proteomic and enzymatic profiles, we show evidence for the induced expression of enzymes and PULs in cells grown on mannan polysaccharides compared with cells grown on glucose. In addition, we show that the secretion of putative biomass-degrading enzymes during growth on glucose comprises a system for nutrient scavenging, which employs constitutively produced enzymes. Significance of this study Chitinophaga pinensis belongs to a bacterial genus which is prominent in microbial communities in agricultural and forest environments, where plant and fungal biomass is intensively degraded. Such degradation is hugely significant in the recycling of carbon in the natural environment, and the enzymes responsible are of biotechnological relevance in emerging technologies involving the deconstruction of plant cell wall material. The bacterium has a comparatively large genome, which includes many uncharacterised carbohydrate-active enzymes. We present the first proteomic assessment of the biomass-degrading machinery of this species, focusing on mannan, an abundant plant cell wall hemicellulose. Our findings include the identification of several novel enzymes, which are promising targets for future biochemical characterisation. In addition, the data indicate the expression of specific Polysaccharide Utilisation Loci, induced in the presence of different growth substrates. We also highlight how a constitutive secretion of enzymes which deconstruct microbial biomass likely forms part of a nutrient scavenging process.

  • 172. Larsbrink, Johan
    et al.
    Tuveng, Tina R.
    Pope, Phillip B.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Eijsink, Vincent G.H.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Proteomic data on enzyme secretion and activity in the bacterium Chitinophaga pinensis2017In: Data in Brief, E-ISSN 2352-3409, Vol. 11, p. 484-490Article in journal (Refereed)
    Abstract [en]

    The secretion of carbohydrate-degrading enzymes by a bacterium sourced from a softwood forest environment has been investigated by mass spectrometry. The findings are discussed in full in the research article “Proteomic insights into mannan degradation and protein secretion by the forest floor bacterium Chitinophaga pinensis” in Journal of Proteomics by Larsbrink et al. ([1], doi: 10.1016/j.jprot.2017.01.003). The bacterium was grown on three carbon sources (glucose, glucomannan, and galactomannan) which are likely to be nutrient sources or carbohydrate degradation products found in its natural habitat. The bacterium was grown on solid agarose plates to mimic the natural behaviour of growth on a solid surface. Secreted proteins were collected from the agarose following trypsin-mediated hydrolysis to peptides. The different carbon sources led to the secretion of different numbers and types of proteins. Most carbohydrate-degrading enzymes were found in the glucomannan-induced cultures. Several of these enzymes may have biotechnological potential in plant cell wall deconstruction for biofuel or biomaterial production, and several may have novel activities. A subset of carbohydrate-active enzymes (CAZymes) with predicted activities not obviously related to the growth substrates were also found in samples grown on each of the three carbohydrates. The full dataset is accessible at the PRIDE partner repository (ProteomeXchange Consortium) with the identifier PXD004305, and the full list of proteins detected is given in the supplementary material attached to this report.

  • 173. Larsson, Mikael
    et al.
    Zhou, Qi
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. KTH, School of Biotechnology (BIO), Glycoscience.
    Larsson, Anette
    Different types of microfibrillated cellulose as filler materials in polysodium acrylate superabsorbents2011In: Chinese Journal of Polymer Science, ISSN 0256-7679, E-ISSN 1439-6203, Vol. 29, no 4, p. 407-413Article in journal (Refereed)
    Abstract [en]

    Three types of microfibrillated cellulose (MFC) with differences in structure and surface charge were used at low concentration as filler materials in polysodium acrylate superabsorbents (SAPs). The swelling of the composite hydrogels was determined in 0.9% NaCl solution as well as in deionized water. The shear modulus of the samples was determined through uniaxial compression analysis after synthesis and after swelling in 0.9% NaCl solution. Furthermore, the ability to retain filler effects after washing was investigated. The results showed that all of the investigated MFCs had a strong reinforcing effect on the shear modulus after synthesis. The filler effect on swelling and on the associated shear modulus of swollen samples showed a more complicated dependence on structure and surface charge. Finally, it was found that the filler effects were reasonably retained after washing and subsequent drying. The results confirm that MFC holds great potential as a filler material in superabsorbent applications. Furthermore, the results provide some insight on how the structural properties and surface charge of MFC will affect gel properties depending on swelling conditions. This information should be useful in evaluating the use of different types of MFC in future applications.

  • 174.
    Le Normand, Myriam
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology.
    Mélida, Hugo
    KTH, School of Biotechnology (BIO), Glycoscience.
    Holmbom, Bjarne
    Åbo Akademi.
    Michaelsen, Terje E.
    Unversity of Oslo.
    Inngjerdingen, Marit
    University of Oslo.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Paulsen, B.S.
    Ek, Monica
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology.
    Hot-water extracts from the inner bark of Norway spruce with immunomodulating activities2014In: Carbohydrate Polymers, ISSN 0144-8617, E-ISSN 1879-1344, Vol. 101, no 1, p. 699-704Article in journal (Refereed)
    Abstract [en]

    The inner bark of Norway spruce (Picea abies) was sequentially extracted with hot water at 100 degrees C, 140 C and 160 degrees C. The hot-water extracts (IB 100 degrees C, IB 140 degrees C and IB 160 degrees C) contained pectic polysaccharides and showed immunostimulating activities. Structural analyses of their carbohydrate content, including glycosidic linkage analyses, revealed the presence of pectins with a large rhamnogalacturonan RG-I domain ramified with highly-branched arabinans. IB 100 degrees C also contained a large amount of terminal glucosyl residues, indicating the presence of highly substituted polymers. IB 160 degrees C was mainly composed of starch. The hot-water extracts were tested for two biological activities, namely complement fixation and macrophage stimulation. IB 100 degrees C exhibited the highest complement fixation activity, with a 1.7-times higher IC(H)50 than the control pectin, while IB 140 degrees C and IB 160 degrees C gave similar IC(H)50 values as the control. Macrophages were stimulated by IB 100 degrees C and IB 140 degrees C in a dose-dependent manner, but not by IB 160 degrees C. IB 100 degrees C presented the highest activity toward macrophages, comparable to the control pectin.

  • 175. Leppänen, Ann-Sofie
    et al.
    Xu, Chunlin
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Parikka, Kirsti
    Eklund, Patrik
    Sjöholm, Rainer
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Tenkanen, Maija
    Willför, Stefan
    Targeted allylation and propargylation of galactose-containing polysaccharides in water2014In: Carbohydrate Polymers, ISSN 0144-8617, E-ISSN 1879-1344, Vol. 100, p. 46-54Article in journal (Refereed)
    Abstract [en]

    Galactose units of spruce galactoglucomannan (GGM), guar galactomannan (GM), and tamarind (galacto)xyloglucan (XG) were selectively allylated. Firstly aldehyde functionalities were formed at the C-6 position via enzymatic oxidation by galactose oxidase. The formed aldehydes were further derivatized by an indium mediated Barbier-Grignard type reaction, resulting in the formation of homoallylic alcohols. In addition to allylic halides, the same reaction procedure was also applicable for GGM, when using propargyl bromide as halide. All reaction steps were done in water, thus the polysaccharides were modified in a one-pot reaction. The formation of the allylated, or propargylated, product was identified by MALDI-TOF-MS. All polysaccharide products were isolated and further characterized by GC-MS or NMR spectroscopy. By this chemo-enzymatic process, we have demonstrated a novel method for derivatization of GGM and other galactose-containing polysaccharides. The derivatized polysaccharides are potential platforms for further functionalizations.

  • 176.
    Liljeblad, Jonathan F. D.
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tyrode, Eric C.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Johnson, Magnus C.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Rutland, Mark W.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Molecular Structure and Stability of Phospholipid Monolayers Probed by Vibrational Sum Frequency Spectroscopy (VSFS)2012In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 102, no 3, p. 591A-591AArticle in journal (Other academic)
  • 177.
    Liljeblad, Jonathan F.D.
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science. KTH, School of Chemical Science and Engineering (CHE), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Rutland, Mark W.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science. KTH, School of Chemical Science and Engineering (CHE), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Johnson, Magnus C.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science. KTH, School of Chemical Science and Engineering (CHE), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Supported Phospholipid Monolayers: The Molecular Structure Investigated by Vibrational Sum Frequency Spectroscopy2011In: The Journal of Physical Chemistry C, ISSN 1932-7447, E-ISSN 1932-7455, Vol. 115, no 21, p. 10617-10629Article in journal (Refereed)
    Abstract [en]

    The molecular structure, packing properties, and hydrating water of Langmuir-Blodgett monolayers of the phospholipids 1,2-distearoyl-sn-glyercophosphatidylcholine (DSPC, 18:0 PC), its deuterated analogue (18:0 PC-d83), and 1,2-distearoyl-sn-glyerco-phosphatidylserine (DSPC, 18:0 PS) deposited on planar calcium fluoride (CaF2) substrates have been investigated using the surface-specific nonlinear optical technique vibrational sum frequency spectroscopy (VSFS). Compression isotherms were recorded before the deposition of the monolayers at a surface pressure of 35 mN/m, mimicking the conditions of biological cell membranes. The CH and CD stretch regions, the water region, and the lower wavenumber region, containing phosphate, ester, carboxylate, and amine signals, thus partly covering the fingerprint region, were probed to obtain a complete map of the molecules. The data indicate that all deposited monolayers formed a well-ordered and stable film, and probing the water region revealed significant differences in hydration for the different headgroups. In addition, the tilt angle of the aliphatic chains relative to the surface normal was estimated to be approximately 4 degrees to 10 degrees based on orientational analysis using the antisymmetric methyl stretching vibration. Orientational analysis of the ester C=O groups was also performed, and the result was consistent with the estimated tilt angle of the aliphatic chains.

  • 178.
    Liljeblad, Jonathan F.D.
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tyrode, Eric
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Rutland, Mark W.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science. Institute for Surface Chemistry, Stockholm, Sweden.
    Johnson, Magnus C.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Phospholipid Monolayers Probed by Vibrational Sum Frequency Spectroscopy: Instability of Unsaturated Phospholipids2010In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 98, p. L50-L52Article in journal (Refereed)
    Abstract [en]

    The surface specific technique vibrational sum frequency spectroscopy has been applied to in situ studies of the degradation of Langmuir monolayers of 1,2-diacyl-phosphocholines with various degrees of unsaturation in the aliphatic chains. To monitor the degradation of the phospholipids, the time-dependent change of the monolayer area at constant surface pressure and the sum frequency intensity of the vinyl CH stretch at the carbon-carbon double bonds were measured. The data show a rapid degradation of monolayers of phospholipids carrying unsaturated aliphatic chains compared to the stable lipids carrying fully saturated chains when exposed to the ambient laboratory air. In addition, the degradation of the phospholipids can be inhibited by purging the ambient air with nitrogen. This instability may be attributed to spontaneous degradation by oxidation mediated by various reactive species in the air. To further elucidate the process of lipid oxidation in biological membranes artificial Langmuir monolayers probed by a surface specific spectroscopic technique as in this study can serve as a model system for studying the degradation/oxidation of cell membrane constituents.

  • 179.
    Liljeblad, Jonathan F.D.
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Rutland, Mark W.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Johnson, C. Magnus
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Stability and structure of phospholipid monolayers probed by vibrational sum frequency spectroscopy (VSFS)2010In: Abstracts of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 239Article in journal (Other academic)
  • 180.
    Liljeblad, Jonathan F.D.
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Rutland, Mark W.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Johnson, Magnus C.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    The Structure of Model Membranes Studied by Vibrational Sum Frequency Spectroscopy2010In: Progress in Colloid and Polymer Science, ISSN 0340-255X, E-ISSN 1437-8027, Vol. 137, p. 9-12Article in journal (Refereed)
    Abstract [en]

    The structure and order of insoluble Langmuir monolayers consisting of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC or 18:0 PC) and the surrounding water molecules have been investigated by vibrational sum frequency spectroscopy (VSFS). At surface pressures of 1, 15, and 57 mN/m corresponding to molecular areas of 53, 50, and 43 Å2, respectively, the DSPC molecules formed a well ordered film. Both the VSF signal from the methyl stretching vibrations of the lipid and the surrounding water increased with enhanced surface pressure, as a result of the higher surface density and increased order of the system. Water molecules hydrating the polar parts of the headgroup and in close contact to the hydrocarbon groups of the lipid were observed in all three polarization combinations of the laser beams, and distinguished by their different vibrational frequencies.

  • 181. Liu, Jun
    et al.
    Kisonen, Victor
    Willfor, Stefan
    Xu, Chunlin
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience.
    Profiling the substitution pattern of xyloglucan derivatives by integrated enzymatic hydrolysis, hydrophilic-interaction liquid chromatography and mass spectrometry2016In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1463, p. 110-120Article in journal (Refereed)
    Abstract [en]

    Plant polysaccharides constitute arguably the most complex family of biomacromolecules in terms of the stereochemistry and regiochemistry of their intramolecular linkages. The chemical modification of such polysaccharides introduces an additional level of complexity for structural determinations. We have developed an integrated analytical procedure combining selective enzymatic hydrolysis, hydrophilic interaction liquid chromatography (HILIC), and mass spectrometry (MS) to describe the substitution pattern of xyloglucan (XyG) and its chemo-enzymatic derivatives (cationic, anionic, and benzyl aminated). Enzymatic hydrolysis of XyG derivatives by a xyloglucan-specific endoglucanase (XEG) generates oligosaccharides amenable for mass spectrometric identification with distinct structures, based on enzymatic substrate recognition and hydrolytic pattern. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) and electrospray ionisation mass spectrometry (ESI-MS) offer qualitative mass profiling of the chemical derivatives. Separation and identification of the complex oligosaccharide profiles released by enzymatic hydrolysis is achieved by hyphenation of hydrophilic interaction liquid chromatography with mass spectrometry (HILIC-ESI-MS). Further fragmentation by tandem mass spectrometry (ESI-MS/MS) in positive mode enables the structural sequencing of modified XyG oligosaccharides and the identification of the substituent position without further derivatisation. This integrated approach can be used to obtain semi-quantitative information of the substitution pattern of hemicellulose derivatives, with fundamental implications for their modification mechanisms and performance.

  • 182. Liu, Yiying
    et al.
    Rzeszutek, Elzbieta
    KTH, School of Biotechnology (BIO), Glycoscience.
    van der Voort, Menno
    Wu, Cheng-Hsuan
    Thoen, Even
    Skaar, Ida
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Dorrestein, Pieter C.
    Raaijmakers, Jos M.
    de Bruijn, Irene
    Diversity of Aquatic Pseudomonas Species and Their Activity against the Fish Pathogenic Oomycete Saprolegnia2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 8, article id e0136241Article in journal (Refereed)
    Abstract [en]

    Emerging fungal and oomycete pathogens are increasingly threatening animals and plants globally. Amongst oomycetes, Saprolegnia species adversely affect wild and cultivated populations of amphibians and fish, leading to substantial reductions in biodiversity and food productivity. With the ban of several chemical control measures, new sustainable methods are needed to mitigate Saprolegnia infections in aquaculture. Here, PhyloChip-based community analyses showed that the Pseudomonadales, particularly Pseudomonas species, represent one of the largest bacterial orders associated with salmon eggs from a commercial hatchery. Among the Pseudomonas species isolated from salmon eggs, significantly more biosurfactant producers were retrieved from healthy salmon eggs than from Saprolegnia-infected eggs. Subsequent in vivo activity bioassays showed that Pseudomonas isolate H6 significantly reduced salmon egg mortality caused by Saprolegnia diclina. Live colony mass spectrometry showed that strain H6 produces a viscosin-like lipopeptide surfactant. This biosurfactant inhibited growth of Saprolegnia in vitro, but no significant protection of salmon eggs against Saprolegniosis was observed. These results indicate that live inocula of aquatic Pseudomonas strains, instead of their bioactive compound, can provide new (micro) biological and sustainable means to mitigate oomycete diseases in aquaculture.

  • 183. Lombard, Vincent
    et al.
    Bernard, Thomas
    Rancurel, Corinne
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Coutinho, Pedro M.
    Henrissat, Bernard
    A hierarchical classification of polysaccharide lyases for glycogenomics2010In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 432, p. 437-444Article in journal (Refereed)
    Abstract [en]

    Carbohydrate-active enzymes face huge substrate diversity in a highly selective manner using only a limited number of available folds. They are therefore subjected to multiple divergent and convergent evolutionary events. This and their frequent modularity render their functional annotation in genomes difficult in a number of cases. In the present paper, a classification of polysaccharide lyases (the enzymes that cleave polysaccharides using an elimination instead of a hydrolytic mechanism) is shown thoroughly for the first time. Based on the analysis of a large panel of experimentally characterized polysaccharide lyases, we examined the correlation of various enzyme properties with the three levels of the classification: fold, family and subfamily. The resulting hierarchical classification, which should help annotate relevant genes in genomic efforts, is available and constantly updated at the Carbohydrate-Active Enzymes Database (http://www.cazy.org).

  • 184.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Glycoscience.
    Allele-specific mutation detection assay2003Patent (Other (popular science, discussion, etc.))
  • 185.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Glycoscience.
    Gene expression analysis in tissue sectionsPatent (Other (popular science, discussion, etc.))
  • 186.
    Lundeberg, Joakim
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Uhlén, Mathias
    Activating a reversibly inactivated immobilized enzyme by release from an immobilizing moiety1995Patent (Other (popular science, discussion, etc.))
  • 187.
    Lundeberg, Joakim
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Uhlén, Mathias
    The use of modular oligonucleotides as probes or primers in nuceic acid based assay1996Patent (Other (popular science, discussion, etc.))
  • 188.
    Lönnberg, Hanna
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Fogelström, Linda
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Malmström, Eva
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Samir, Said
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Berglund, Lars
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Hult, Anders
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    POLY 661-Grafting of poly(e-caprolactone) from microfibrillated cellulose films: for biocomposite applications2007Conference paper (Refereed)
  • 189.
    Lönnberg, Hanna
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Fogelström, Linda
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Hult, Anders
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Berglund, Lars
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Malmström, Eva
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Investigation of the graft length impact on the interfacial toughness in a cellulose/poly(ε-caprolactone) bilayer laminate2011In: Composites Science And Technology, ISSN 0266-3538, E-ISSN 1879-1050, Vol. 71, no 1, p. 9-12Article in journal (Refereed)
    Abstract [en]

    Interfacial adhesion between immiscible cellulose–polymer interfaces is a crucial property for fibrous biocomposites. To tailor the interfacial adhesion, the grafting of polymers from cellulose films was studied using ring-opening polymerization of ε-caprolactone. The poly(ε-caprolactone) (PCL) grafted cellulose was analyzed with FTIR, AFM and via water CA measurements. The graft length was varied by the addition of a free initiator, enabling tailoring of the interfacial toughness. Films of microfibrillated cellulose were grafted with PCL and hot-pressed together with a PCL-film to form a bilayer laminate. Interfacial peeling toughness correlates very strongly with PCL degree of polymerization (DP). PCL grafts form physical entanglements in the PCL matrix and promote significant plastic deformation in the PCL bulk, thus increasing interfacial peeling energy.

  • 190.
    Malm, Erik
    KTH, School of Biotechnology (BIO), Glycoscience.
    Analyzing the properties and biosynthesis of β-glucans from Gluconacetobacter and poplar2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Glucans are polysaccharides integral to many materials and biological functions. Under the umbrella of Biomime, the Swedish Center for Biomimetic Fiber Engineering, this work has aimed to improve basic understanding of the biosynthesis of such glucans. This has been achieved through direct investigation of cellulose structure, and by developing the tools to analyze glucan biosynthesis. Notably we have identified a novel chemical effector of glucan synthesis processes and developed a proteomic toolkit useful for analyzing membrane-bound glycosyltransferases, the enzyme group responsible for glucan biosynthesis. During this work, glucan synthesis has been studied using both Gluconacetobacter and Populus cell suspension cultures.

    Publication I. Gluconacetobacter cellulose (BC) was used as a base to create a novel and well characterized nano-material with improved mechanical properties. This novel composite of BC and hydroxyethylcellulose (HEC) had improved tensile strength compared to pure BC. Through thorough study utilizing dispersion measurements, electron microscopy, nuclear magnetic resonance and X-ray diffraction it was shown that the improved properties derived from a layer of HEC coating each fibril.

    Publication II. Bacterial cellulose was labeled in specific positions with 13C (C4 and C6). These samples were analyzed by CP/MAS NMR along with cellulose samples from cotton and Halocynthia sp. For each sample spectral fitting was performed and general properties of crystal allomorph composition and fibril widths were determined. Calculations were also made for water accessible surfaces of the fibrils. The results showed that water accessible C4 surface signals are reflective of the allomorph composition of the sample, along with a distorted signal that derives due to fibril imperfections. Water accessible surface signals from the C6 region are instead derived from rotamer conformations of the C6 hydroxymethyl groupsfrom glucose residues.

    In Publication III, a high-throughput screen was used to identify an inhibitor of Golgi-derived glycosyltransferase activity, termed chemical A. The structural basis for inhibition was determined and in vitro assays of callose synthesis were performed. The in vitro assays revealed chemical A to also be an activator of callose synthesis. To understand this activation kinetic studies were performed, showing that chemical A is a mixed type of activator, which can bind either the free enzyme or the enzyme-substrate complex. Chemical A has uses in chemical genetics for dissecting processes involving callose synthesis, such as stress response and cell-plate formation.

    In publication IV, we present an in-house developed platform for proteomics with a distributed processing model. This in-house system has been central to many proteomics tasks, including for those presented in publication V, and is being distributed as the Automated Proteomics Pipeline (APP).

    In publication V, conditions for enrichment of Detergent-Resistant Microdomains (DRM) have been optimized for Populus trichocarpa cell cultures. The proteins enriched in DRM were identified using mass spectrometry based proteomics, and a functional model for DRM was proposed. This model involves proteins specialized in stress response, including callose synthase, and cell signaling. This further strengthens the arguments for DRMs as sites of specific cellular functions and confirms they play a role in glucan synthesis.

  • 191.
    Malm, Erik
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Wickholm, Kristina
    Larsson, Per Tomas
    Iversen, Tommy
    The surface structure of well-ordered native cellulose fibrils in contact with water2010In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 345, no 1, p. 97-100Article in journal (Refereed)
    Abstract [en]

    CP/MAS C-13 NMR spectroscopy was used in combination with spectral fitting to examine the surface structure of hydrated cellulose I fibrils from Halocynthia and Gluconoacetobacter xylinus. To increase the spectral intensities and minimize signal overlap, G. xylinus celluloses site-specifically enriched in C-13 either on C4 or on both C1 and C6 were examined. The experimental data showed multiple C4 and C6 signals for the water accessible fibril surfaces in the highly crystalline celluloses. These signal multiplicities were attributed to structural features in the surface layers induced by the fibril interior, and could not be extracted by spectral fitting in celluloses with a lower degree of crystallinity such as cellulose from cotton.

  • 192.
    Malm, Erik
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Sundqvist, Gustav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    APP: An Automated Proteomics Pipeline for the analysis of mass spectrometry data based on multiple open access tools2014In: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 15, no 1, article id 345Article in journal (Refereed)
    Abstract [en]

    Background: Mass spectrometry analyses of complex protein samples yield large amounts of data and specific expertise is needed for data analysis, in addition to a dedicated computer infrastructure. Furthermore, the identification of proteins and their specific properties require the use of multiple independent bioinformatics tools and several database search algorithms to process the same datasets. In order to facilitate and increase the speed of data analysis, there is a need for an integrated platform that would allow a comprehensive profiling of thousands of peptides and proteins in a single process through the simultaneous exploitation of multiple complementary algorithms. Results: We have established a new proteomics pipeline designated as APP that fulfills these objectives using a complete series of tools freely available from open sources. APP automates the processing of proteomics tasks such as peptide identification, validation and quantitation from LC-MS/MS data and allows easy integration of many separate proteomics tools. Distributed processing is at the core of APP, allowing the processing of very large datasets using any combination of Windows/Linux physical or virtual computing resources. Conclusions: APP provides distributed computing nodes that are simple to set up, greatly relieving the need for separate IT competence when handling large datasets. The modular nature of APP allows complex workflows to be managed and distributed, speeding up throughput and setup. Additionally, APP logs execution information on all executed tasks and generated results, simplifying information management and validation.

  • 193. Marcotuli, Ilaria
    et al.
    Hsieh, Yves S. -Y.
    KTH, School of Biotechnology (BIO), Glycoscience. University of Adelaide, Australia.
    Lahnstein, Jelle
    Yap, Kuok
    Burton, Rachel Anita
    Blanco, Antonio
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fincher, Geoffrey Bruce
    Gadaleta, Agata
    Structural Variation and Content of Arabinoxylans in Endosperm and Bran of Durum Wheat (Triticum turgidum L.)2016In: Journal of Agricultural and Food Chemistry, ISSN 0021-8561, E-ISSN 1520-5118, Vol. 64, no 14, p. 2883-2892Article in journal (Refereed)
    Abstract [en]

    Arabinoxylans are one group of dietary fiber components in cereal grains, and specific health benefits have been linked with their molecular fine structures and hence with physicochemical properties such as solubility in aqueous media. To characterize the fiber quality for functional foods, starchy endosperm and bran fractions from 11 durum wheat lines were analyzed for total and water-soluble arabinoxylans, (1,3;1,4)-beta-glucan, and bound ferulic acid. The arabinoxylan contents ranged from 11 to 16.4% (w/w) in bran and from 1.5 to 1.8% in the starchy endosperm. Of the starchy endosperm arabinoxylans, 37% was soluble in water. No correlation was found between arabinoxylan content and bound ferulic acid in bran, although a relatively high level of this antioxidant was found in endosperm (38.3 mu g/g endosperm flour). Enzymatic fingerprinting was performed to define the major fine structural features of arabinoxylans from both regions of the grain. Five major oligosaccharides released by xylanase hydrolysis were identified and characterized in the 11 durum lines. In addition, DP5, DP6, and DP7 oligosaccharides containing five, six, and seven pentosyl residues, respectively, were purified.

  • 194. Maris, An
    et al.
    Kaewthai, Nomchit
    KTH, School of Biotechnology (BIO), Glycoscience.
    Eklöf, Jens
    KTH, School of Biotechnology (BIO), Glycoscience.
    Miller, Janice G.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fry, Stephen C.
    Verbelen, Jean-Pierre
    Vissenberg, Kris
    Differences in enzymic properties of five recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis thaliana2011In: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 62, no 1, p. 261-271Article in journal (Refereed)
    Abstract [en]

    Xyloglucan endotransglucosylase/hydrolases (XTHs) are cell wall enzymes that are able to graft xyloglucan chains to oligosaccharides or to other available xyloglucan chains and/or to hydrolyse xyloglucan chains. As they are involved in the modification of the load-bearing cell-wall components, they are believed to be very important in the regulation of growth and development. Given the large number (33) of XTH genes in Arabidopsis and the overlapping expression patterns, specific enzymic properties may be expected. Five predominantly root-expressed Arabidopsis thaliana XTHs belonging to subgroup I/II were analysed here. These represent two sets of closely related genes: AtXTH12 and 13 on the one hand (trichoblast-enriched) and AtXTH17, 18, and 19 on the other (expressed in nearly all cell types in the root). They were all recombinantly produced in the yeast Pichia pastoris and partially purified by ammonium sulphate precipitation before they were subsequently all subjected to a series of identical in vitro tests. The kinetic properties of purified AtXTH13 were investigated in greater detail to rule out interference with the assays by contaminating yeast proteins. All five proteins were found to exhibit only the endotransglucosylase (XET; EC 2.4.1.207) activity towards xyloglucan and non-detectable endohydrolytic (XEH; EC 3.2.1.151) activity. Their endotransglucosylase activity was preferentially directed towards xyloglucan and, in some cases, water-soluble cellulose acetate, rather than to mixed-linkage beta-glucan. Isoforms differed in optimum pH (5.0-7.5), in temperature dependence and in acceptor substrate preferences.

  • 195.
    Mark, Pekka
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Baumann, Martin J.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Eklöf, Jens M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Gullfot, Fredrika
    KTH, School of Biotechnology (BIO), Glycoscience.
    Michel, Gurvan
    Kallas, Åsa M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Czjzek, Mirjam
    Analysis of nasturtium TmNXG1 complexes by crystallography and molecular dynamics provides detailed insight into substrate recognition by family GH16 xyloglucan endo-transglycosylases and endo-hydrolases2009In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 75, no 4, p. 820-836Article in journal (Refereed)
    Abstract [en]

    Reorganization and degradation of the wall crosslinking and seed storage polysaccharide xyloglucan by glycoside hydrolase family 16 (GH16) endo-transglycosylases and hydrolases are crucial to the growth of the majority of land plants, affecting processes as diverse as germination, morphogenesis, and fruit ripening. A high-resolution, three-dimensional structure of a nasturtium (Tropaeolum majus) endo-xyloglucanase loop mutant, TmNXG1-Delta YNIIG, with an ohgosaccharide product bound in the negative active-site subsites, has been solved by X-ray crystallography. Comparison of this novel complex to that of the strict xyloglucan endotransglycosylase PttXET16-34 from hybrid aspen (Populus tremula x tremuloides), previously solved with a xylogluco-oligosaccharide bound in the positive subsites, highlighted key protein structures that affect the disparate catalytic activities displayed by these closely related enzymes. Combination of these "partial" active-site complexes through molecular dynamics simulations in water allowed modeling of wild-type TmNXG1, TmNXG1-Delta YNIIG, and wild-type PttXET16-34 in complex with a xyloglucan octadecasaccharide spanning the entire catalytic cleft. A comprehensive analysis of these full-length complexes underscored the importance of various loops lining the active site. Subtle differences leading to a tighter hydrogen bonding pattern on the negative (glycosyl donor) binding subsites, together with loop flexibility on the positive (glycosyl acceptor) binding subsites appear to favor hydrolysis over transglycosylation in GH16 xyloglucan-active enzymes.

  • 196.
    Mark, Pekka
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Zhang, Qiong
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Czjzek, Mirjam
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ågren, Hans
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Molecular dynamics simulations of a branched tetradecasaccharide substrate in the active site of a xyloglucan endo-transglycosylase2011In: Molecular Simulation, ISSN 0892-7022, E-ISSN 1029-0435, Vol. 37, no 12, p. 1001-1013Article in journal (Refereed)
    Abstract [en]

    Molecular dynamics simulations of the tetradecasaccharide XXXGXXXG in complex with the hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 have been performed and analysed with respect to structure, dynamics, flexibility and ligand interactions. Notably, the charge state of the so-called 'helper residue' aspartate 87 (Asp87), which lies between the catalytic nucleophile [glutamate 85 (Glu85)] and general acid/base (Glu89) residues on the same beta strand, had a significant effect on PttXET16-34 active site structure. When Asp87 was deprotonated, electrostatic repulsion forced the nucleophile away from C1 of the sugar ring in subsite - 1 and the proton-donating ability of Glu89 was also weakened due to the formation of a hydrogen bond with Asp87, whereas the protonation of Asp87 resulted in the formation of a hydrogen bond with the catalytic nucleophile and correct positioning of the catalytic machinery. The results suggest that catalysis in glycoside hydrolase family 16, and by extension clan GH-B enzymes, is optimal when the catalytic nucleophile is deprotonated for nucleophilic attack on the substrate, whereas the 'helper residue' and general acid/base residue are both in their conjugate acid forms to align the nucleophile and deliver a proton to the departing sugar, respectively.

  • 197.
    Mark, Pekka
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Zhang, Qiong
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Czjzek, Mirjam
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ågren, Hans
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Molecular dynamics simulations of a branched tetradecasaccharide substrate in the active site of a xyloglucan endo-transglycosylaseIn: Molecular Simulation, ISSN 0892-7022, E-ISSN 1029-0435Article in journal (Refereed)
    Abstract [en]

    Molecular dynamics (MD) simulations of the tetradecasaccharide XXXGXXXG in complex with the hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 have been performed and analyzed with respect to structure, dynamics, flexibility and ligand interactions. Notably, the charge state of the so-called “helper residue” Asp87, which lies between the catalytic nucleophile (Glu85) and general acid/base (Glu89) residues on the same beta strand, had a significant effect on PttXET16-34 active site structure. When Asp87 was deprotonated, electrostatic repulsion forced the nucleophile Glu85 away from C-1 of the sugar ring in subsite -1 and the electrophile Glu89 was also weakened due to the formation of a hydrogen bond to Asp87, whereas the protonation of Asp87 resulted in the formation of a hydrogen bond with the catalytic nucleophile and correct positioning of the catalytic machinery. The results suggest that catalysis in glycoside hydrolase family 16, and by extension clan GH-B enzymes, is optimal when the catalytic nucleophile is deprotonated for nucleophilic attack on the substrate, while the “helper residue” and general acid/base residue are both in their conjugate-acid forms to align the nucleophile and deliver a proton to the departing sugar, respectively.

  • 198.
    Martinez-Abad, Antonio
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Berglund, Jennie
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Toriz, Guillermo
    Gatenholm, Paul
    Henriksson, Gunnar
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Lindström, Mikael
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Wohlert, Jakob
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Regular Motifs in Xylan Modulate Molecular Flexibility and Interactions with Cellulose Surfaces2017In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 175, no 4, p. 1579-1592Article in journal (Refereed)
    Abstract [en]

    Xylan is tightly associated with cellulose and lignin in secondary plant cell walls, contributing to its rigidity and structural integrity in vascular plants. However, the molecular features and the nanoscale forces that control the interactions among cellulose microfibrils, hemicelluloses, and lignin are still not well understood. Here, we combine comprehensive mass spectrometric glycan sequencing and molecular dynamics simulations to elucidate the substitution pattern in softwood xylans and to investigate the effect of distinct intramolecular motifs on xylan conformation and on the interaction with cellulose surfaces in Norway spruce (Picea abies). We confirm the presence of motifs with evenly spaced glycosyl decorations on the xylan backbone, together with minor motifs with consecutive glucuronation. These domains are differently enriched in xylan fractions extracted by alkali and subcritical water, which indicates their preferential positioning in the secondary plant cell wall ultrastructure. The flexibility of the 3-fold screw conformation of xylan in solution is enhanced by the presence of arabinofuranosyl decorations. Additionally, molecular dynamic simulations suggest that the glycosyl substitutions in xylan are not only sterically tolerated by the cellulose surfaces but that they increase the affinity for cellulose and favor the stabilization of the 2-fold screw conformation. This effect is more significant for the hydrophobic surface compared with the hydrophilic ones, which demonstrates the importance of nonpolar driving forces on the structural integrity of secondary plant cell walls. These novel molecular insights contribute to an improved understanding of the supramolecular architecture of plant secondary cell walls and have fundamental implications for overcoming lignocellulose recalcitrance and for the design of advanced wood-based materials.

  • 199. Martinez-Fleites, Carlos
    et al.
    Guerreiro, Catarina I. P. D.
    Baumann, Martin J.
    KTH, School of Biotechnology (BIO).
    Taylor, Edward J.
    Prates, Jose A. M.
    Ferreira, Luis M. A.
    Fontes, Carlos M. G. A.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Davies, Gideon J.
    Crystal structures of Clostridium thermocellum xyloglucanase, XGH74A, reveal the structural basis for xyloglucan recognition and degradation2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 34, p. 24922-24933Article in journal (Refereed)
    Abstract [en]

    The enzymatic degradation of the plant cell wall is central both to the natural carbon cycle and, increasingly, to environmentally friendly routes to biomass conversion, including the production of biofuels. The plant cell wall is a complex composite of cellulose microfibrils embedded in diverse polysaccharides collectively termed hemicelluloses. Xyloglucan is one such polysaccharide whose hydrolysis is catalyzed by diverse xyloglucanases. Here we present the structure of the Clostridium thermocellum xyloglucanase Xgh74A in both apo and ligand-complexed forms. The structures, in combination with mutagenesis data on the catalytic residues and the kinetics and specificity of xyloglucan hydrolysis reveal a complex subsite specificity accommodating seventeen monosaccharide moieties of the multibranched substrate in an open substrate binding terrain.

  • 200.
    Martínez-Abad, Antonio
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ruthes, Andrea C.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Enzymatic-assisted extraction and modification of lignocellulosic plant polysaccharides for packaging applications2015In: Journal of Applied Polymer Science, ISSN 0021-8995, E-ISSN 1097-4628, Vol. 133, no 2, article id 42523Article in journal (Refereed)
    Abstract [en]

    Plant polysaccharides comprise the main renewable resource available in the biosphere for biomaterial production. However, the recalcitrant and heterogeneous structure of lignocellulosic biomass hinders the effective fractionation and exploitation of the polysaccharide components for the design of carbohydrate-based materials. Carbohydrate-active enzymes constitute a selective and versatile biotechnological tool that can assist during the biomass pretreatment steps to extract and fractionate the polysaccharide macromolecular components. Moreover, this enzymatic toolbox can be as well exploited for the tailored modification of the molecular structure of relatively pure polysaccharide components to achieve customized macroscopic properties. This review critically discusses the potential and challenges of the use of plant lignocellulosic polysaccharides and enzymatic modifications to design and prepare suitable materials for packaging applications in terms of their structure-property relations. Structural factors such as the molar mass and crystallinity of the polysaccharide fractions and functional factors such as water sensitivity and processability of the derived films are critical for the material performance.

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