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  • 51. Butchosa, Nria
    et al.
    Brown, Christian
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Berglund, Lars A.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites. 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.
    Antimicrobial activity of biocomposites based on bacterial cellulose and chitin nanoparticles2012In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 243Article in journal (Other academic)
  • 52.
    Butchosa, Nuria
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Brown, Christian
    KTH, School of Biotechnology (BIO), Glycoscience.
    Larsson, Per Tomas
    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, Biocomposites. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Nanocomposites of bacterial cellulose nanofibers and chitin nanocrystals: fabrication, characterization and bactericidal activity2013In: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 15, no 12, p. 3404-3413Article in journal (Refereed)
    Abstract [en]

    An environmentally friendly approach was implemented for the production of nanocomposites with bactericidal activity, using bacterial cellulose (BC) nanofibers and chitin nanocrystals (ChNCs). The antibacterial activity of ChNCs prepared by acid hydrolysis, TEMPO-mediated oxidation or partial deacetylation of a-chitin powder was assessed and the structure of the ChNC nanoparticles was characterized by X-ray diffraction, atomic force microscopy, and solid-state C-13-NMR. The partially deacetylated ChNCs (D-ChNC) showed the strongest antibacterial activity, with 99 +/- 1% inhibition of bacterial growth compared to control samples. Nanocomposites were prepared from BC nanofibers and D-ChNC by (i) in situ biosynthesis with the addition of D-ChNC nanoparticles in the culture medium of Acetobacter aceti, and (ii) post-modification by mixing D-ChNC with disintegrated BC in an aqueous suspension. The structure and mechanical properties of the BC/D-ChNC nanocomposites were characterized by Fourier transform infrared spectroscopy, elemental analysis, field-emission scanning electron microscopy, and an Instron universal testing machine. The bactericidal activity of the nanocomposites increased with the D-ChNC content, with a reduction in bacterial growth by 3.0 log units when the D-ChNC content was 50%. D-ChNC nanoparticles have great potential as substitutes for unfriendly antimicrobial compounds such as heavy metal nanoparticles and synthetic polymers to introduce antibacterial properties to cellulosic materials.

  • 53.
    Butchosa, Nuria
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Leijon, Felicia
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Enhancing toughness of cellulose nanofibrils through the expression of cellulose-binding modules in plantManuscript (preprint) (Other academic)
  • 54.
    Butchosa, Nuria
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Water redispersible nanofibrillated cellulose adsorbed with carboxymethyl cellulose2014In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 247, p. 130-CELL-Article in journal (Other academic)
  • 55.
    Butchosa, Núria
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Water redispersible cellulose nanofibrils adsorbed with carboxymethyl cellulose2014In: Cellulose (London), ISSN 0969-0239, E-ISSN 1572-882X, Vol. 21, no 6, p. 4349-4358Article in journal (Refereed)
    Abstract [en]

    Cellulose nanofibrils (CNFs) are difficult to redisperse in water after they have been completely dried due to the irreversible agglomeration of cellulose during drying. Here, we have developed a simple process to prepare water-redispersible dried CNFs by the adsorption of small amounts of carboxymethyl cellulose (CMC) and oven drying. The adsorption of CMC onto CNFs in water suspensions at 22 and 121 °C was studied, and the adsorbed amount of CMC was measured via conductimetric titration. The water-redispersibility of dried CNFs adsorbed with different amounts of CMC was characterized by sedimentation test. Above a critical threshold of CMC adsorption, i.e. 2.3 wt%, the oven dried CNF–CMC sample was fully redispersible in water. The morphology, rheological, and mechanical properties of water-redispersed CNF–CMC samples were investigated by field emission scanning electron microscopy, viscosity measurement, and tensile test, respectively. The water-redispersed CNFs preserved the original properties of never dried CNFs. This new method will facilitate the production, transportation and storage, and large-scale industrial applications of CNFs.

  • 56. Bygdell, Joakim
    et al.
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Obudulu, Ogonna
    Srivastava, Manoj K.
    Nilsson, Robert
    Sundberg, Bjorn
    Trygg, Johan
    Mellerowicz, Ewa J.
    Wingsle, Gunnar
    Protein expression in tension wood formation monitored at high tissue resolution in Populus2017In: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 68, no 13, p. 3405-3417Article in journal (Refereed)
    Abstract [en]

    Tension wood (TW) is a specialized tissue with contractile properties that is formed by the vascular cambium in response to gravitational stimuli. We quantitatively analysed the proteomes of Populus tremula cambium and its xylem cell derivatives in stems forming normal wood (NW) and TW to reveal the mechanisms underlying TW formation. Phloem-, cambium-, and wood-forming tissues were sampled by tangential cryosectioning and pooled into nine independent samples. The proteomes of TW and NW samples were similar in the phloem and cambium samples, but diverged early during xylogenesis, demonstrating that reprogramming is an integral part of TW formation. For example, 14-3-3, reactive oxygen species, ribosomal and ATPase complex proteins were found to be up-regulated at early stages of xylem differentiation during TW formation. At later stages of xylem differentiation, proteins involved in the biosynthesis of cellulose and enzymes involved in the biosynthesis of rhamnogalacturonan-I, rhamnogalacturonan-II, arabinogalactan-II and fasciclin-like arabinogalactan proteins were up-regulated in TW. Surprisingly, two isoforms of exostosin family proteins with putative xylan xylosyl transferase function and several lignin biosynthesis proteins were also up-regulated, even though xylan and lignin are known to be less abundant in TW than in NW. These data provided new insight into the processes behind TW formation.

  • 57. Cantu-Jungles, Thaisa Moro
    et al.
    Ruthes, Andrea Caroline
    KTH, School of Biotechnology (BIO), Glycoscience. Universidade Federal do Paraná, Brazil.
    El-Hindawy, Marwa
    Moreno, Roberta Barbara
    Zhang, Xiaowei
    Cordeiro, Lucimara M. C.
    Hamaker, Bruce R.
    Iacomini, Marcello
    In vitro fermentation of Cookeina speciosa glucans stimulates the growth of the butyrogenic Clostridium cluster XIVa in a targeted way2018In: Carbohydrate Polymers, ISSN 0144-8617, E-ISSN 1879-1344, Vol. 183, p. 219-229Article in journal (Refereed)
    Abstract [en]

    Dietary fiber chemical and physical structures may be critical to the comprehension of how they may modulate gut bacterial composition. We purified insoluble polymers from Cookeina speciosa, and investigated its fermentation profile in an in vitro human fecal fermentation model. Two glucans, characterized as a (1 -> 3),(1 -> 6)-linked and a (1 -> 3)-linked beta-D-glucans were obtained. Both glucans were highly butyrogenic and propiogenic, with low gas production, during in vitro fecal fermentation and led to distinct bacterial shifts if compared to fructooligosaccharides. Specific increases in Bacteroides uniformis and genera from the Clostridium cluster XIVa, such as butyrogenic Anaerostipes and Roseburia were observed. The (1 -> 3)-linked beta-D-glucan presented a faster fermentation profile compared to the branched (1 -> 3),(1 -> 6)-linked beta-D-glucan. Our findings support the view that depending on its fine chemical structure, and likely its insoluble nature, these dietary fibers can be utilized to direct a targeted promotion of the intestinal microbiota to butyrogenic Clostridium cluster XIVa bacteria.

  • 58. Cartmell, Alan
    et al.
    McKee, Lauren
    KTH, School of Biotechnology (BIO). University of Georgia, United States.
    Pena, Maria J.
    Larsbrink, Johan
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kaneko, Satoshi
    Ichinose, Hitomi
    Lewis, Richard J.
    Vikso-Nielsen, Anders
    Gilbert, Harry J.
    Marles-Wright, Jon
    The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 17Article in journal (Refereed)
    Abstract [en]

    Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo-and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific alpha-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave alpha-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific alpha-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed beta-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for alpha-1,2-L-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.

  • 59. Castro, A.
    et al.
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience.
    Nilsson, L.
    Characterization of a water soluble, hyperbranched arabinogalactan from yacon (Smallanthus sonchifolius) roots2017In: Food Chemistry, ISSN 0308-8146, E-ISSN 1873-7072, Vol. 223, p. 76-81Article in journal (Refereed)
    Abstract [en]

    Yacon (Smallanthus sonchifolius Poepp. & Endl.) roots are largely grown in Andean countries and have attracted recent interest due to their antioxidant and prebiotic effects. Yacon is typically consumed as a fruit due to its sweet taste and juiciness. The macromolecular properties of an aqueous extract of yacon are investigated using asymmetric flow field-flow fractionation (AF4) coupled to UV, multiangle light scattering (MALS) and differential refractive index (dRI) detection. The method allows for determination of molar mass and size over the size distribution. Three major populations were found of which one strongly dominates in concentration. Through collection of fractions from AF4, carbohydrate composition and glycosidic linkage analysis for the dominating population was performed. The results show that the dominating population consists of a highly branched arabinogalactan (type 2) with a molar mass of approximately 1–2 · 105 g/mol, a hydrodynamic radius of approximately 6–10 nm and a relatively high apparent density (approx. 70–150 kg/m3).

  • 60. Cho, Sung Hyun
    et al.
    Purushotham, Pallinti
    Fang, Chao
    Maranas, Cassandra
    Diaz-Moreno, Sara M
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zimmer, Jochen
    Kumar, Manish
    Nixon, B. Tracy
    Synthesis and Self-Assembly of Cellulose Microfibrils from Reconstituted Cellulose Synthase2017In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 175, no 1, p. 146-156Article in journal (Refereed)
    Abstract [en]

    Cellulose, the major component of plant cell walls, can be converted to bioethanol and is thus highly studied. In plants, cellulose is produced by cellulose synthase, a processive family-2 glycosyltransferase. In plant cell walls, individual beta-1,4-glucan chains polymerized by CesA are assembled into microfibrils that are frequently bundled into macrofibrils. An in vitro system in which cellulose is synthesized and assembled into fibrils would facilitate detailed study of this process. Here, we report the heterologous expression and partial purification of His-tagged CesA5 from Physcomitrella patens. Immunoblot analysis and mass spectrometry confirmed enrichment of PpCesA5. The recombinant protein was functional when reconstituted into liposomes made from yeast total lipid extract. The functional studies included incorporation of radiolabeled Glc, linkage analysis, and imaging of cellulose microfibril formation using transmission electron microscopy. Several microfibrils were observed either inside or on the outer surface of proteoliposomes, and strikingly, several thinner fibrils formed ordered bundles that either covered the surfaces of proteoliposomes or were spawned from liposome surfaces. We also report this arrangement of fibrils made by proteoliposomes bearing CesA8 from hybrid aspen. These observations describe minimal systems of membrane-reconstituted CesAs that polymerize beta-1,4-glucan chains that coalesce to form microfibrils and higher-ordered macrofibrils. How these micro-and macrofibrils relate to those found in primary and secondary plant cell walls is uncertain, but their presence enables further study of the mechanisms that govern the formation and assembly of fibrillar cellulosic structures and cell wall composites during or after the polymerization process controlled by CesA proteins.

  • 61. Cifuentes, Carolina
    et al.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Emons, Anne Mie C.
    Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitro2010In: J INTEGR PLANT BIOL, ISSN 1672-9072, Vol. 52, no 2, p. 221-233Article in journal (Refereed)
    Abstract [en]

    The conditions that favor the in vitro synthesis of cellulose from tobacco BY-2 cell extracts were determined. The procedure leading to the highest yield of cellulose consisted of incubating digitonin extracts of membranes from 11-day-old tobacco BY-2 cells in the presence of 1 mM UDP-glucose, 8 mM Ca2+ and 8 mM Mg2+. Under these conditions, up to nearly 40% of the polysaccharides synthesized in vitro corresponded to cellulose, the other polymer synthesized being callose. Transmission electron microscopy analysis revealed the occurrence of two types of structures in the synthetic reactions. The first type consisted of small aggregates with a diameter between 3 and 5 nm that associated to form fibrillar strings of a maximum length of 400 nm. These structures were sensitive to the acetic/nitric acid treatment of Updegraff and corresponded to callose. The second type of structures was resistant to the Updegraff reagent and corresponded to straight cellulose microfibrils of 2-3 nm in diameter and 200 nm to up to 5 mu m in length. In vitro reactions performed on electron microscopy grids indicated that the minimal rate of microfibril elongation in vitro is 120 nm/min. Measurements of retardance by liquid crystal polarization microscopy as a function of time showed that small groups of microfibrils increased in retardance by up to 0.047 nm/min per pixel, confirming the formation of organized structures.

  • 62. Collins, Catherine M.
    et al.
    Murray, Patrick G.
    Denman, Stuart
    Morrissey, John P.
    Byrnes, Lucy
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tuohy, Maria G.
    Molecular cloning and expression analysis of two distinct beta-glucosidase genes, bg1 and aven1, with very different biological roles from the thermophilic, saprophytic fungus Talaromyces emersonii2007In: Mycological Research, ISSN 0953-7562, E-ISSN 1469-8102, Vol. 111, p. 840-849Article in journal (Refereed)
    Abstract [en]

    Recent sequencing of a number of fungal genomes has revealed the presence of multiple putative beta-glucosidases. Here, we report the cloning of two beta-glucosidase genes (bg1 and aven1), which have very different biological functions and represent two of a number of beta-glucosidases from Talaromyces emersonii. The bg1 gene, encoding a putative intracellular beta-glucosidase, shows significant similarity to other fungal glucosidases from glycosyl hydrolase family 1, known to be involved in cellulose degradation. Solka floc, methyl-xylose, gentiobiose, beech wood xylan, and lactose induced expression of bg1, whereas glucose repressed expression. A second beta-glucosidase gene isolated from T. emersonii, aueni, encodes a putative avenacinase, an enzyme that deglucosylates the anti-fungal saponin, avenacin, rendering it less toxic to the fungus. This gene displays high homology with other fungal saponin-hydrolysing enzymes and beta-glucosidases within GH3. A putative secretory signal peptide of 21 amino acids was identified at the N-terminus of the predicted aven1 protein sequence suggesting that this enzyme is extracellular. Furthermore, T. emersonii cultivated on oat plant biomass was shown to deglucosylate avenacin. The presence of the avenacinase transcript was confirmed by RT-PCR on RNA extracted from mycelia grown in the presence of avenacin. The expression pattern of aven1 on various carbon sources was distinctly different from that of bg1. Only methyl-xylose and gentiobiose induced transcription of aven1. Gentiobiose induces synthesis of a number of cellulase genes by T. emersonii and it may be a possible candidate for the natural cellulase inducer observed in Penicillium purpurogenum. This work represents the first report of an avenacinase gene from a thermophilic, saprophytic fungal source, and suggests that this gene is not exclusive to plant pathogens.

  • 63. Comfort, Donald A.
    et al.
    Bobrov, Kirill S.
    Ivanen, Dina R.
    Shabalin, Konstantin A.
    Harris, James M.
    Kulminskaya, Anna A.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kelly, Robert M.
    Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 11, p. 3319-3330Article in journal (Refereed)
    Abstract [en]

    Organization of glycoside hydrolase (GH) families into clans expands the utility of information on catalytic mechanisms of member enzymes. This issue was examined for GH27 and GH36 through biochemical analysis of GH36 alpha-galactosidase from Thermotoga maritima (TmGalA). Catalytic residues in TmGalA were inferred through structural homology with GH27 members to facilitate design of site-directed mutants. Product analysis confirmed that the wild type (WT) acted with retention of anomeric stereochemistry, analogous to GH27 enzymes. Conserved acidic residues were confirmed through kinetic analysis of D327G and D387G mutant enzymes, azide rescue, and determination of azide rescue products. Mutation of Asp327 to Gly resulted in a mutant that had a 200-800-fold lower catalytic rate on aryl galactosides relative to the WT enzyme. Azide rescue experiments using the D327G enzyme showed a 30-fold higher catalytic rate compared to without azide. Addition of azide to the reaction resulted in formation of azide beta-D-galactopyranoside, confirming Asp327 as the nucleophilic residue. The Asp387Gly mutation was 1500-fold catalytically slower than the WT enzyme on p-nitrophenyl alpha-D-galactopyranoside. Analysis at different pH values produced a bell-shaped curve of the WT enzyme, but D387G exhibited higher activity with increasing pH. Catalyzed reactions with the D387G mutant in the presence of azide resulted in formation of azide alpha-D-galactopryanoside as the product of a retaining mechanism. These results confirm that Asp387 is the acid/base residue of TmGalA. Furthermore, they show that the biochemical characteristics of GH36 TmGalA are closely related to GH27 enzymes, confirming the mechanistic commonality of clan GH-D members.

  • 64.
    Cunha, Ana Gisela
    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.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Larsson, Per Tomas
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. INNVENTIA AB, Sweden.
    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.
    Topochemical acetylation of cellulose nanopaper structures for biocomposites: mechanisms for reduced water vapour sorption2014In: Cellulose (London), ISSN 0969-0239, E-ISSN 1572-882X, Vol. 21, no 4, p. 2773-2787Article in journal (Refereed)
    Abstract [en]

    Moisture sorption decreases dimensional stability and mechanical properties of polymer matrix biocomposites based on plant fibers. Cellulose nanofiber reinforcement may offer advantages in this respect. Here, wood-based nanofibrillated cellulose (NFC) and bacterial cellulose (BC) nanopaper structures, with different specific surface area (SSA), ranging from 0.03 to 173.3 m(2)/g, were topochemically acetylated and characterized by ATR-FTIR, XRD, solid-state CP/MAS C-13-NMR and moisture sorption studies. Polymer matrix nanocomposites based on NFC were also prepared as demonstrators. The surface degree of substitution (surface-DS) of the acetylated cellulose nanofibers is a key parameter, which increased with increasing SSA. Successful topochemical acetylation was confirmed and significantly reduced the moisture sorption in nanopaper structures, especially at RH = 53 %. BC nanopaper sorbed less moisture than the NFC counterpart, and mechanisms are discussed. Topochemical NFC nanopaper acetylation can be used to prepare moisture-stable nanocellulose biocomposites.

  • 65.
    Dahlin, Paul
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. Stockholm Univ, Sweden.
    Müller, Marion C.
    KTH, School of Biotechnology (BIO), Glycoscience. Stockholm Univ, Sweden.
    Ekengren, Sophia
    KTH, School of Biotechnology (BIO), Glycoscience. Stockholm Univ, Sweden.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, Australia.
    The Impact of Steroidal Glycoalkaloids on the Physiology of Phytophthora infestans, the Causative Agent of Potato Late Blight2017In: Molecular Plant-Microbe Interactions, ISSN 0894-0282, E-ISSN 1943-7706, Vol. 30, no 7, p. 531-542Article in journal (Refereed)
    Abstract [en]

    Steroidal glycoalkaloids (SGAs) are plant secondary metabolites known to be toxic to animals and humans and that have putative roles in defense against pests. The proposed mechanisms of SGA toxicity are sterol-mediated disruption of membranes and inhibition of cholinesterase activity in neurons. It has been suggested that phytopathogenic microorganisms can overcome SGA toxicity by enzymatic deglycosylation of SGAs. Here, we have explored SGA-mediated toxicity toward the invasive oomycete Phytophthora infestans, the causative agent of the late blight disease in potato and tomato, as well as the potential for SGA deglycosylation by this species. Our growth studies indicate that solanidine, the nonglycosylated precursor of the potato SGAs a-chaconine and a-solanine, has a greater physiological impact than its glycosylated forms. All of these compounds were incorporated into the mycelium, but only solanidine could strongly inhibit the growth of P. infestans in liquid culture. Genes encoding several glycoside hydrolases with potential activity on SGAs were identified in the genome of P. infestans and were shown to be expressed. However, we found no indication that deglycosylation of SGAs takes place. We present additional evidence for apparent host-specific adaptation to potato SGAs and assess all results in terms of future pathogen management strategies.

  • 66.
    Dahlin, Paul
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. Stockholm University, Sweden.
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. University of Adelaide, Australia.
    Mckee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    The Oxidosqualene Cyclase from the Oomycete Saprolegnia parasitica Synthesizes Lanosterol as a Single Product2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, article id 1802Article in journal (Refereed)
    Abstract [en]

    The first committed step of sterol biosynthesis is the cyclisation of 2,3-oxidosqualene to form either lanosterol (LA) or cycloartenol (CA). This is catalyzed by an oxidosqualene cyclase (OSC). LA and CA are subsequently converted into various sterols by a series of enzyme reactions. The specificity of the OSC therefore determines the final composition of the end sterols of an organism. Despite the functional importance of OSCs, the determinants of their specificity are not well understood. In sterol-synthesizing oomycetes, recent bioinformatics, and metabolite analysis suggest that LA is produced. However, this catalytic activity has never been experimentally demonstrated. Here, we show that the OSC of the oomycete Saprolegnia parasitica, a severe pathogen of salmonid fish, has an uncommon sequence in a conserved motif important for specificity. We present phylogenetic analysis revealing that this sequence is common to sterol-synthesizing oomycetes, as well as some plants, and hypothesize as to the evolutionary origin of some microbial sequences. We also demonstrate for the first time that a recombinant form of the OSC from S. parasitica produces LA exclusively. Our data pave the way for a detailed structural characterization of the protein and the possible development of specific inhibitors of oomycete OSCs for disease control in aquaculture.

  • 67.
    Dahlin, Paul
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ekengren, Sophia
    KTH, School of Biotechnology (BIO), Glycoscience.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Comparative analysis of sterol acquisition in the oomycetes Saprolegnia parasitica and Phytophthora infestans2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 2, article id e0170873Article in journal (Refereed)
    Abstract [en]

    The oomycete class includes pathogens of animals and plants which are responsible for some of the most significant global losses in agriculture and aquaculture. There is a need to replace traditional chemical means of controlling oomycete growth with more targeted approaches, and the inhibition of sterol synthesis is one promising area. To better direct these efforts, we have studied sterol acquisition in two model organisms: the sterol-autotrophic Saprolegnia parasitica, and the sterol-heterotrophic Phytophthora infestans. We first present a comprehensive reconstruction of a likely sterol synthesis pathway for S. parasitica, causative agent of the disease saprolegniasis in fish. This pathway shows multiple potential routes of sterol synthesis, and draws on several avenues of new evidence: bioinformatic mining for genes with sterol-related functions, expression analysis of these genes, and analysis of the sterol profiles in mycelium grown in different media. Additionally, we explore the extent to which P. infestans, which causes the late blight in potato, can modify exogenously provided sterols. We consider whether the two very different approaches to sterol acquisition taken by these pathogens represent any specific survival advantages or potential drug targets.

  • 68. Daniel, Geoffrey
    et al.
    Filonova, Lada
    Kallas, Asa M.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Morphological and chemical characterisation of the G-layer in tension wood fibres of Populus tremula and Betula verrucosa: Labelling with cellulose-binding module CBM1(HjCel7A) and fluorescence and FE-SEM microscopy2006In: Holzforschung, ISSN 0018-3830, E-ISSN 1437-434X, Vol. 60, no 6, p. 618-624Article in journal (Refereed)
    Abstract [en]

    The gelatinous layer (G-layer) formed on the lumen wall in early- and latewood fibres of poplar and birch tension wood was characterised using a novel molecular marker specific for crystalline cellulose in conjunction with fluorescence and FE-SEM microscopy. Crystalline cellulose was localised using a cloned Cel7A cellulose-binding module (CBM1(HjCel7A)) from the fungus Hypocrea jecorina conjugated directly to FITC/TRITC or indirectly via a secondary antibody conjugated to FITC for fluorescence microscopy or to gold/silver for FE-SEM. With the CBM1(HjCel7A) conjugate, the G-layer was clearly distinguished from other secondary cell-wall layers as a bright green layer visible in fibres of tension wood in fluorescence microscopy. FEM-SEM images revealed the supramolecular architecture of the G-layer of poplar wood, which consists of well-defined, often concentrically orientated, cellulose aggregates of the order of 30-40 nm. The cellulose aggregates typically have a microfibril angle of almost 0 degrees. Studies on cellulose marked with CBM1(HjCel7A) followed by Au labelling and Ag enhancement complemented the fluorescence observations. The studies demonstrate the usefulness of this novel molecular marker for crystalline cellulose in situ, which was previously difficult to localise. Further proof of distinct cellulose aggregates was observed.

  • 69. Derba-Maceluch, Marta
    et al.
    Awano, Tatsuya
    Takahashi, Junko
    Lucenius, Jessica
    Ratke, Christine
    Kontro, Inkeri
    Busse-Wicher, Marta
    Kosik, Ondrej
    Tanaka, Ryo
    Winzell, Anders
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kallas, Åsa
    KTH, School of Biotechnology (BIO), Glycoscience.
    Lesniewska, Joanna
    Berthold, Fredrik
    Immerzeel, Peter
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Dupree, Paul
    Serimaa, Ritva
    Mellerowicz, Ewa J.
    Suppression of xylan endotransglycosylase PtxtXyn10A affects cellulose microfibril angle in secondary wall in aspen wood2015In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 205, no 2, p. 666-681Article in journal (Refereed)
    Abstract [en]

    Certain xylanases from family GH10 are highly expressed during secondary wall deposition, but their function is unknown. We carried out functional analyses of the secondary-wall specific PtxtXyn10A in hybrid aspen (Populus tremulaxtremuloides).PtxtXyn10A function was analysed by expression studies, overexpression in Arabidopsis protoplasts and by downregulation in aspen.PtxtXyn10A overexpression in Arabidopsis protoplasts resulted in increased xylan endotransglycosylation rather than hydrolysis. In aspen, the enzyme was found to be proteolytically processed to a 68kDa peptide and residing in cell walls. Its downregulation resulted in a corresponding decrease in xylan endotransglycosylase activity and no change in xylanase activity. This did not alter xylan molecular weight or its branching pattern but affected the cellulose-microfibril angle in wood fibres, increased primary growth (stem elongation, leaf formation and enlargement) and reduced the tendency to form tension wood. Transcriptomes of transgenic plants showed downregulation of tension wood related genes and changes in stress-responsive genes. The data indicate that PtxtXyn10A acts as a xylan endotransglycosylase and its main function is to release tensional stresses arising during secondary wall deposition. Furthermore, they suggest that regulation of stresses in secondary walls plays a vital role in plant development.

  • 70. Dimitroff, George
    et al.
    Little, Alan
    Lahnstein, Jelle
    Schwerdt, Julian G.
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    Burton, Rachel A.
    Fincher, Geoffrey B.
    (1,3;1,4)-beta-Glucan Biosynthesis by the CSLF6 Enzyme: Position and Flexibility of Catalytic Residues Influence Product Fine Structure2016In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, no 13, p. 2054-2061Article in journal (Refereed)
    Abstract [en]

    Cellulose synthase-like F6 (CslF6) genes encode polysaccharide synthases responsible for (1,3;1,4)-beta-glucan biosynthesis in cereal grains. However, it is not clear how both (1,3)- and (1,4) -linkages are incorporated into a single polysaccharide chain and how the frequency and arrangement of the two linkage types that define the fine structure of the polysaccharide are controlled. Through transient expression in Nicotiana benthamiana leaves, two CSLF6 orthologs from different cereal species were shown to mediate the synthesis of (1,3;1,4)-beta-glucans with very different fine structures. Chimeric cDNA constructs with interchanged sections of the barley and sorghum CslF6 genes were developed to identify regions of the synthase enzyme responsible for these differences. A single amino acid residue upstream of the TED motif in the catalytic region was shown to dramatically change the fine structure of the polysaccharide produced. The structural basis of this effect can be rationalized by reference to a homology model of the enzyme and appears to be related to the position and flexibility of the TED motif in the active site of the enzyme. The region and amino acid residue identified provide opportunities to manipulate the solubility of (1,3;1,4)-beta-glucan in grains and vegetative tissues of the grasses and, in particular, to enhance the solubility of dietary fibers that are beneficial to human health.

  • 71.
    Djerbi, Soraya
    KTH, School of Biotechnology (BIO), Glycoscience.
    Cellulose synthases in Populus- identification, expression analyses and in vitro synthesis2005Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Cellulose is a biopolymer of great relevance in the plant cell walls, where it constitutes the most important skeletal component. Cellulose is also an important raw material in the pulp- and paper, forest, and textile industries, among others. Cellulose biosynthesis in particular, and xylogenesis in general are processes which are currently poorly understood. Yet, research in cellulose synthesis is progressing and different applications of cellulose, mainly cellulose derivatives for e.g. pharmaceuticals and coatings, are constantly emerging. This thesis depicts how cellulose synthase (CesA) genes in Populus were identified and characterized by gene expression- and bioinformatics analyses. Within an EST database of more than 100,000 clones from wood forming tissues of three different Populus taxa, ten CesA genes were identified in Populus tremula x tremuloides. Subsequent gene expression analyses by using microarrays and real-time PCR experiments in woody tissues, revealed distinct regulation patterns among the genes of interest. This enabled proper classification and characterization of the secondary cell wall related CesA genes, in particular. Bioinformatic analyses of the genome sequence of Populus trichocarpa further provided a complete picture of the number of putative CesA genes retained after several duplication events during tree evolution. In contrast to the previously reported set of ten 'true' CesA genes in many other plant species, the genome of P. trichocarpa encodes 18 putative proteins, which could be assembled into nine groups according to their sequence similarities. Interestingly, studies in the EST database suggested that paralogs within at least two groups have corresponding orthologs in P. tremula x tremuloides, which are furthermore transcribed. This implies that at least some of the duplicated genes have remained functional, or may have acquired a modified function.

    By focusing on the CesA genes associated with secondary cell wall formation, cellulose synthesis was also studied in poplar cell suspension cultures. Selection of CesA enriched material was performed by determining expression intensities of the CesA genes using RT-PCR, whereupon membrane protein extraction was initiated. CesA proteins are part of large cellulose synthesizing complexes in the plasma membrane. Subsequent proteomic approaches comprised partial purification of these cellulose synthesizing complexes from protein enriched culture material and in vitro cellulose synthesis experiments. De novo synthesized material was successfully characterized and the acquired yields were as high as 50% cellulose (compared to previously reported yields of 30% in other plant systems) of the total in vitro synthesized product. Elevated CesA gene expression levels can thus be correlated to increased protein activity in poplar cell suspension cultures. In addition, antibodies raised against CesA antigens were used in Western blot analyses comprising samples along the protein extraction- and purification procedure. Proteins with corresponding molecular weight to the theoretical 120kDa of CesA proteins were recognized by a range of different specific antibodies. The study demonstrates that poplar cell suspension cultures can provide a valuable model system for studies of cellulose synthesis and different aspects of xylogenesis.

  • 72. Doliška, A.
    et al.
    Willför, S.
    Strnad, S.
    Ribitsch, V.
    Kleinschek, K. S.
    Eklund, P.
    Xu, Chunlin
    KTH, School of Biotechnology (BIO), Glycoscience.
    Antithrombotic properties of sulfated wood-derived galactoglucomannans2012In: Holzforschung, ISSN 0018-3830, E-ISSN 1437-434X, Vol. 66, no 2, p. 149-154Article in journal (Refereed)
    Abstract [en]

    Galactoglucomannans (GGMs) are water-soluble polysaccharides released and accumulated in process waters in the production of thermomechanical pulp. The general trend in the forestry industry is moving towards bio-refineries, for example utilizing these hemicelluloses as bioactive substances. At present, there is no industrial use of wood-derived mannans. In this study, GGMs extracted from thermomechanical pulp, as well as further carboxymethyl-ated galactoglucomannans (CM-GGMs), were sulfated to increase their antithrombotic properties. The products were characterized with Fourier transform infrared spectroscopy, nuclear magnetic resonance and capillary electrophoresis. The carbohydrate composition and sulfur amounts were determined. The products' total charges were determined by polyelectrolyte titrations and their antithrombotic effect was measured based on the activated partial thromboplastin time. The results showed a significant increase in the antithrombotic effect of the sulfated galactoglucomannans from spruce wood and thus a potential new use for wood-derived mannans.

  • 73. Douchkov, D.
    et al.
    Lueck, S.
    Hensel, G.
    Kumlehn, J.
    Rajaraman, J.
    Johrde, A.
    Doblin, M. S.
    Beahan, C. T.
    Kopischke, M.
    Fuchs, R.
    Lipka, V.
    Niks, R. E.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, Australia.
    Chowdhury, J.
    Little, A.
    Burton, R. A.
    Bacic, A.
    Fincher, G. B.
    Schweizer, P.
    The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus2016In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 212, no 2, p. 421-433Article in journal (Refereed)
    Abstract [en]

    Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively.

  • 74.
    Duval, Antoine
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Vilaplana, Francisco
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. KTH, School of Biotechnology (BIO), Glycoscience.
    Crestini, Claudia
    Lawoko, Martin
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Solvent screening for the fractionation of industrial kraft lignin2016In: Holzforschung, ISSN 0018-3830, E-ISSN 1437-434X, Vol. 70, no 1, p. 11-20Article in journal (Refereed)
    Abstract [en]

    The polydispersity of commercially available kraft lignins (KLs) is one of the factors limiting their applications in polymer-based materials. A prerequisite is thus to develop lignin fractionation strategies compatible with industrial requirements and restrictions. For this purpose, a solvent-based lignin fractionation technique has been addressed. The partial solubility of KL in common industrial solvents compliant with the requirements of sustainable chemistry was studied, and the results were discussed in relation to Hansen solubility parameters. Based on this screening, a solvent sequence is proposed, which is able to separate well-defined KL fractions with low polydispersity.

  • 75.
    Eklof, Jens M.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. Michael Smith Laboratories, Department of Chemistry, University of British Columbia, Canada.
    Ruda, Marcus C.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. Michael Smith Laboratories, Department of Chemistry, University of British Columbia, Canada.
    Distinguishing xyloglucanase activity in endo-β(1 → 4)glucanases2012In: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 510, p. 97-120Article in journal (Refereed)
    Abstract [en]

    The ability of beta-glucanases to cleave xyloglucans, a family of highly decorated beta-glucans ubiquitous in plant biomass, has traditionally been overlooked in functional biochemical studies. An emerging body of data indicates, however, that a spectrum of xyloglucan specificity resides in diverse glycoside hydrolases from a range of carbohydrate-active enzyme families including classic "cellulase" families. This chapter outlines a series of enzyme kinetic and product analysis methods to establish degrees of xyloglucan specificity and modes of action of glycosidases emerging from enzyme discovery projects.

  • 76. Eklund, D. Magnus
    et al.
    Staldal, Veronika
    Valsecchi, Isabel
    Cierlik, Izabela
    Eriksson, Caitriona
    Hiratsu, Keiichiro
    Ohme-Takagi, Masaru
    Sundstrom, Jens F.
    Thelander, Mattias
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Sundberg, Eva
    The Arabidopsis thaliana STYLISH1 Protein Acts as a Transcriptional Activator Regulating Auxin Biosynthesis2010In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 22, no 2, p. 349-363Article in journal (Refereed)
    Abstract [en]

    The establishment and maintenance of auxin maxima in vascular plants is regulated by auxin biosynthesis and polar intercellular auxin flow. The disruption of normal auxin biosynthesis in mouse-ear cress ( Arabidopsis thaliana) leads to severe abnormalities, suggesting that spatiotemporal regulation of auxin biosynthesis is fundamental for normal growth and development. We have shown previously that the induction of the SHORT-INTERNODES/STYLISH (SHI/STY) family member STY1 results in increased transcript levels of the YUCCA (YUC) family member YUC4 and also higher auxin levels and auxin biosynthesis rates in Arabidopsis seedlings. We have also shown previously that SHI/STY family members redundantly affect development of flowers and leaves. Here, we further examine the function of STY1 by analyzing its DNA and protein binding properties. Our results suggest that STY1, and most likely other SHI/STY members, are DNA binding transcriptional activators that target genes encoding proteins mediating auxin biosynthesis. This suggests that the SHI/STY family members are essential regulators of auxin-mediated leaf and flower development. Furthermore, the lack of a shoot apical meristem in seedlings carrying a fusion construct between STY1 and a repressor domain, SRDX, suggests that STY1, and other SHI/STY members, has a role in the formation and/or maintenance of the shoot apical meristem, possibly by regulating auxin levels in the embryo.

  • 77.
    Eklöf, Jens
    KTH, School of Biotechnology (BIO), Glycoscience.
    A holistic approach to understanding CAZy families through reductionist methods2009Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

     

    In a time when the amount of biological data present in the public domain is becoming increasingly vast, the need for good classification systems has never been greater. In the field of glycoscience the necessity of a good classification for the enzymes involved in the biosynthesis, modification and degradation of polysaccharides is even more pronounced than in other fields. This is due to the complexity of the substrates, the polysaccharides, as the theoretical number of possible hexa-oligosaccharides from only hexoses exceeds 1012 isomers! 

    An initiative to classify enzymes acting on carbohydrates began around 1990 by the French scientist Bernard Henrissat. The resulting database, the Carbohydrate Active enzymes database (CAZy), classifies enzymes by sequence similarity into families allowing the inference of structure and catalytic mechanism. What CAZy does not provide however, are means to understand how members of a family are related, and in what way they differ from each other. The top-down approach used in this thesis, combining phylogenetic analysis of whole CAZy families, or sub-families, with structural determinations and detailed kinetic analysis allows for exactly that.  

    Finding determinants for transglycosylation versus hydrolysis within the xth gene product family of GH16 as well as restricting the hydrolytic enzymes to a well defined clade are integral parts of paper I. In paper II a new bacterial sub-clade within CE8 was discovered. The structural determination of theEscherichia coli outer membrane lipoprotein YbhC from from the new sub-clade explained the difference in specificity. The information provided in the two papers of this thesis gives a better understanding of the development of different specificities of diverse CAZY families as well as it aids in future gene product annotations. Furthermore this work has begun to fill the white spots uncovered in the phylogenetic trees.

     

     

  • 78.
    Eklöf, Jens
    KTH, School of Biotechnology (BIO), Glycoscience.
    Plant and microbial xyloglucanases: Function, Structure and Phylogeny2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In this thesis, enzymes acting on the primary cell wall hemicellulose xyloglucan are studied.  Xyloglucans are ubiquitous in land plants which make them an important polysaccharide to utilise for microbes and a potentially interesting raw material for various industries.  The function of xyloglucans in plants is mainly to improve primary cell wall characteristics by coating and tethering cellulose microfibrils together.  Some plants also utilise xyloglucans as storage polysaccharides in their seeds.

    In microbes, a variety of different enzymes for degrading xyloglucans have been found.  In this thesis, the structure-function relationship of three different microbial endo-xyloglucanases from glycoside hydrolase families 5, 12 and 44 are probed and reveal details of the natural diversity found in xyloglucanases.  Hopefully, a better understanding of how xyloglucanases recognise and degrade their substrate can lead to improved saccharification processes of plant matter, finding uses in for example biofuel production.

    In plants, xyloglucans are modified in muro by the xyloglucan transglycosylase/hydrolase (XTH) gene products.  Interestingly, closely related XTH gene products catalyse either transglycosylation (XET activity) or hydrolysis (XEH activity) with dramatically different effects on xyloglucan and on cell wall characteristics.  The strict transglycosylases transfer xyloglucan segments between individual xyloglucan molecules while the hydrolases degrade xyloglucan into oligosaccharides.  Here, we describe and determine, a major determinant of transglycosylation versus hydrolysis in XTH gene products by solving and comparing the first 3D structure of an XEH, Tm-NXG1 and a XET, PttXET16-34.  The XEH activity was hypothesised, and later confirmed to be restricted to subset of the XTH gene products.  The in situ localisation of XEH activity in roots and hypocotyls of Arabidopsis was also visualised for the first time.  Furthermore, an evolutionary scheme for how XTH gene products developed from bacterial beta-1,3;1,4 glucanases was also presented based on the characterisation of a novel plant endo-glucanase, PtEG16-1. The EG16s are proposed to predate XTH gene products and are with activity on both xyloglucan and beta-1,3;1,4 glucans an “intermediate” in the evolution from beta-1,3;1,4 glucanases to XTH gene products.

  • 79.
    Eklöf, Jens
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    An endo β‐1,4 glucanse, PtEG16‐1 from black cottonwood (Populustrichocarpa) represents an evolutionary link between bacterial lichenases and XTH geneproductsArticle in journal (Other academic)
  • 80.
    Eklöf, Jens M.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    The XTH Gene Family: An Update on Enzyme Structure, Function, and Phylogeny in Xyloglucan Remodeling2010In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 153, no 2, p. 456-466Article in journal (Refereed)
  • 81.
    Eklöf, Jens M.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Shojania, S.
    Okon, M.
    McIntosh, L. P.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Structure-function analysis of a broad specificity Populus trichocarpa endo-β-glucanase reveals an evolutionary link between bacterial licheninases and plant XTH gene products2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 22, p. 15786-15799Article in journal (Refereed)
    Abstract [en]

    The large xyloglucan endotransglycosylase/hydrolase (XTH) gene family continues to be the focus of much attention in studies of plant cell wall morphogenesis due to the unique catalytic functions of the enzymes it encodes. The XTH gene products compose a subfamily of glycoside hydrolase family 16 (GH16), which also comprises a broad range of microbial endoglucanases and endogalactanases, as well as yeast cell wall chitin/β-glucan transglycosylases. Previous whole-family phylogenetic analyses have suggested that the closest relatives to the XTH gene products are the bacterial licheninases (EC 3.2.1.73), which specifically hydrolyze linear mixed linkage β(1→3)/β(1→4)-glucans. In addition to their specificity for the highly branched xyloglucan polysaccharide, XTH gene products are distinguished from the licheninases and other GH16 enzyme subfamilies by significant active site loop alterations and a large C-terminal extension. Given these differences, the molecular evolution of the XTH gene products in GH16 has remained enigmatic. Here, we present the biochemical and structural analysis of a unique, mixed function endoglucanase from black cottonwood (Populus trichocarpa), which reveals a small, newly recognized subfamily of GH16 members intermediate between the bacterial licheninases and plant XTH gene products. We postulate that this clade comprises an important link in the evolution of the large plant XTH gene families from a putative microbial ancestor. As such, this analysis provides new insights into the diversification of GH16 and further unites the apparently disparate members of this important family of proteins. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

  • 82.
    Eklöf, Jens M.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO).
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    The crystal structure of the outer membrane lipoprotein YbhC from Escherichia coli sheds new light on the phylogeny of carbohydrate esterase family 82009In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 76, no 4, p. 1029-1036Article in journal (Refereed)
  • 83. Eneyskaya, E. V.
    et al.
    Ivanen, D. R.
    Shabalin, K. A.
    Kulminskaya, A. A.
    Backinowsky, L. V.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Neustroev, K. N.
    Chemo-enzymatic synthesis of 4-methylumbelliferyl beta-(1 -> 4)-D-xylooligosides: new substrates for beta-D-xylanase assays2005In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 3, no 1, p. 146-151Article in journal (Refereed)
    Abstract [en]

    Transglycosylation catalyzed by a beta-D-xylosidase from Aspergillus sp. was used to synthesize a set of 4-methylumbelliferyl (MU) beta-1-->4-D-xylooligosides having the common structure [beta-D-Xyl-(1-->4)](2-5)-beta- D-Xyl-MU. MU xylobioside synthesized chemically by the condensation of protected MU beta-D-xylopyranoside with ethyl 2,3,4-tri-O-acetyl-1-thio-beta-D-xylopyranoside was used as a substrate for transglycosylation with the beta-D- xylosidase from Aspergillus sp. to produce higher MU xylooligosides. The structures of oligosaccharides obtained were established by H-1 and C-13 NMR spectroscopy and electrospray tandem mass spectrometry. MU beta-D-xylooligosides synthesized were tested as fluorogenic substrates for the GH-10 family beta-D-xylanase from Aspergillus orizae and the GH-11 family beta-D- xylanase I from Trichoderma reesei. Both xylanases released the aglycone from MU xylobioside and the corresponding trioside. With substrates having d.p. 4 and 5, the enzymes manifested endolytic activities, splitting off MU, MUX, and MUX2 primarily.

  • 84. Eneyskaya, Elena V.
    et al.
    Sundqvist, Gustav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Golubev, Alexander M.
    Ibatullin, Farid M.
    Ivanen, Dina R.
    Shabalin, Konstantin A.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kulminskaya, Anna A.
    Transglycosylating and hydrolytic activities of the beta-mannosidase from Trichoderma reesei2009In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 91, no 5, p. 632-638Article in journal (Refereed)
    Abstract [en]

    A purified beta-mannosidase (EC 3.2.1.25) from the fungus Trichoderma reesei has been identified as a member of glycoside hydrolase family 2 through mass spectrometry analysis of tryptic peptides. In addition to hydrolysis, the enzyme catalyzes substrate transglycosylation with p-nitrophenyl beta-mannopyranoside. Structures of the major and minor products of this reaction were identified by NMR analysis as p-nitrophenyl mannobiosides and p-nitrophenyl mannotriosides containing beta-(1 -> 4) and beta-(1 -> 3) linkages. The rate of donor substrate hydrolysis increased in presence of acetonitrile and dimethylformamide, while transglycosylation was weakly suppressed by these organic solvents. Differential ultraviolet spectra of the protein indicate that a rearrangement of the hydrophobic environment of the active site following the addition of the organic solvents may be responsible for this hydrolytic activation.

  • 85. Escudero, Viviana
    et al.
    Jorda, Lucia
    Sopena-Torres, Sara
    Melida, Hugo
    Miedes, Eva
    Munoz-Barrios, Antonio
    Swami, Sanjay
    Alexander, Danny
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Sanchez-Vallet, Andrea
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Jones, Alan M.
    Molina, Antonio
    Alteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the beta-subunit of the heterotrimeric G-protein2017In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 92, no 3, p. 386-399Article in journal (Refereed)
    Abstract [en]

    Arabidopsis heterotrimeric G-protein complex modulates pathogen-associated molecular pattern-triggered immunity (PTI) and disease resistance responses to different types of pathogens. It also plays a role in plant cell wall integrity as mutants impaired in the G- (agb1-2) or G-subunits have an altered wall composition compared with wild-type plants. Here we performed a mutant screen to identify suppressors of agb1-2 (sgb) that restore susceptibility to pathogens to wild-type levels. Out of the four sgb mutants (sgb10-sgb13) identified, sgb11 is a new mutant allele of ESKIMO1 (ESK1), which encodes a plant-specific polysaccharide O-acetyltransferase involved in xylan acetylation. Null alleles (sgb11/esk1-7) of ESK1 restore to wild-type levels the enhanced susceptibility of agb1-2 to the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM), but not to the bacterium Pseudomonas syringae pv. tomato DC3000 or to the oomycete Hyaloperonospora arabidopsidis. The enhanced resistance to PcBMM of the agb1-2 esk1-7 double mutant was not the result of the re-activation of deficient PTI responses in agb1-2. Alteration of cell wall xylan acetylation caused by ESK1 impairment was accompanied by an enhanced accumulation of abscisic acid, the constitutive expression of genes encoding antibiotic peptides and enzymes involved in the biosynthesis of tryptophan-derived metabolites, and the accumulation of disease resistance-related secondary metabolites and different osmolites. These esk1-mediated responses counterbalance the defective PTI and PcBMM susceptibility of agb1-2 plants, and explain the enhanced drought resistance of esk1 plants. These results suggest that a deficient PTI-mediated resistance is partially compensated by the activation of specific cell-wall-triggered immune responses. Significance Statement The plant heterotrimeric G protein complex is an essential component of Pathogen Associated Molecular Pattern-triggered immunity (PTI) and of plant disease resistance to several types of pathogens. We found that modification of the degree of xylan acetylation in plant cell walls activates PTI-independent resistance responses that counterbalance the hypersusceptibility to particular pathogens of plants lacking the heterotrimeric G subunit. These data demonstrate that immune deficient response can be partially compensated by the activation of cell wall-triggered immunity that confers specific disease resistance.

  • 86.
    Ezcurra, Ines
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Johansson, Camilla
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tamizhselvan, Prashanth
    KTH, School of Biotechnology (BIO), Glycoscience.
    Winzell, Anders
    KTH, School of Biotechnology (BIO), Glycoscience.
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Glycoscience.
    An AC-type element mediates transactivation of secondary cell wall carbohydrate-active enzymes by PttMYB021, the Populus MYB46 orthologue2011In: BMC Proceedings, ISSN 1753-6561, E-ISSN 1753-6561, Vol. 5Article in journal (Refereed)
  • 87.
    Ezekiel Mushi, Ngesa
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Butchosa, Núria
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. 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, Biocomposites. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Nanopaper membranes from chitin-protein composite nanofibers: Structure and mechanical properties2014In: Journal of Applied Polymer Science, ISSN 0021-8995, E-ISSN 1097-4628, Vol. 131, no 7, p. 40121-Article in journal (Refereed)
    Abstract [en]

    Chitin nanofibers may be of interest as a component for nanocomposites. Composite nanofibers are therefore isolated from crab shells in order to characterize structure and analyze property potential. The mechanical properties of the porous nanopaper structures are much superior to regenerated chitin membranes. The nanofiber filtration-processing route is much more environmentally friendly than for regenerated chitin. Minerals and extractives are removed using HCl and ethanol, respectively, followed by mild NaOH treatment and mechanical homogenization to maintain chitin-protein structure in the nanofibers produced. Atomic force microscope (AFM) and scanning transmission electron microscope (STEM) reveal the structure of chitin-protein composite nanofibers. The presence of protein is confirmed by colorimetric method. Porous nanopaper membranes are prepared by simple filtration in such a way that different nanofiber volume fractions are obtained: 43%, 52%, 68%, and 78%. Moisture sorption isotherms, structural properties, and mechanical properties of membranes are measured and analyzed. The current material is environmentally friendly, the techniques employed for both individualization and membrane preparation are simple and green, and the results are of interest for development of nanomaterials and biocomposites.

  • 88.
    Ezekiel Mushi, Ngesa
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Joby Kochumalayil, Jose
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Cervin, Nicholas
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. 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, Biocomposites. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Nanostructured hydrogel based on small diameter native chitin nanofibers: Preparation, structure and propertiesManuscript (preprint) (Other academic)
  • 89.
    Ezekiel Mushi, Ngesa
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Nurani, Ghasem
    KTH, School of Biotechnology (BIO), Glycoscience.
    Utsel, Simon
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. 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, Biocomposites. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Soft, bio-inspired chitin/protein nanocomposites: mechanical behavior and interface interactions between recombinant resilin-like proteins and chitin nanofibersManuscript (preprint) (Other academic)
  • 90.
    Fernandes, Susana C. M.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. University of the Basque Country, Spain.
    Alonso-Varona, Ana
    Palomares, Teodoro
    Zubillaga, Veronica
    Labidi, Jalel
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. University of Adelaide, Australia.
    Exploiting Mycosporines as Natural Molecular Sunscreens for the Fabrication of UV-Absorbing Green Materials2015In: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 7, no 30, p. 16558-16564Article in journal (Refereed)
    Abstract [en]

    Ultraviolet radiations have many detrimental effects in living organisms that challenge the stability and function of cellular structures. UV exposure also alters the properties and durability of materials and affects their lifetime. It is becoming increasingly important to develop new biocompatible and environmentally friendly materials to address these issues. Inspired by the strategy developed by fish, algae, and microorganisms exposed to UV radiations in confined ecosystems, we have constructed novel UV-protective materials that exclusively consist of natural compounds. Chitosan was chosen as the matrix for grafting mycosporines and mycosporine-like amino acids as the functional components of the active materials. Here, we show that these materials are biocompatible, photoresistant, and thermoresistant, and exhibit a highly efficient absorption of both UV-A and UV-B radiations. Thus, they have the potential to provide an efficient protection against both types of UV radiations and overcome several shortfalls of the current UV-protective products. In practice, the same concept can be applied to other biopolymers than chitosan and used to produce multifunctional materials. Therefore, it has a great potential to be exploited in a broad range of applications in living organisms and nonliving systems.

  • 91.
    Fernandes, Susana C. M.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, ARC Ctr Excellence Plant Cell Walls, Urrbrae, SA, Australia.
    UV-absorbing materials based on natural molecular sunscreens and chitosan2017In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 253Article in journal (Other academic)
  • 92. Fink, Helen
    et al.
    Ahrenstedt, Lage
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bodin, Aase
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Gatenholm, Paul
    Krettek, Alexandra
    Risberg, Bo
    Bacterial cellulose modified with xyloglucan bearing the adhesion peptide RGD promotes endothelial cell adhesion and metabolism - a promising modification for vascular grafts2011In: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, Vol. 5, no 6, p. 454-463Article in journal (Refereed)
    Abstract [en]

    Today, biomaterials such as polytetrafluorethylene (ePTFE) are used clinically as prosthetic grafts for vascular surgery of large vessels (>5 mm). In small diameter vessels, however, their performance is poor due to early thrombosis. Bacterial-derived cellulose (BC) is a new promising material as a replacement for blood vessels. This material is highly biocompatible in vivo but shows poor cell adhesion. In the native blood vessel, the endothelium creates a smooth non-thrombogenic surface. In order to sustain cell adhesion, BC has to be modified. With a novel xyloglucan (XG) glycoconjugate method, it is possible to introduce the cell adhesion peptide RGD (Arg-Gly-Asp) onto bacterial cellulose. The advantage of the XG-technique is that it is an easy one-step procedure carried out in water and it does not weaken or alter the fiber structure of the hydrogel. In this study, BC was modified with XG and XGRGD to asses primary human vascular endothelial cell adhesion, proliferation, and metabolism as compared with unmodified BC. This XG-RGD-modification significantly increased cell adhesion and the metabolism of seeded primary endothelial cells as compared with unmodified BC whereas the proliferation rate was affected only to some extent. The introduction of an RGD-peptide to the BC surface further resulted in enhanced cell spreading with more pronounced stress fiber formation and mature phenotype. This makes BC together with the XG-method a promising material for synthetic grafts in vascular surgery and cardiovascular research.

  • 93. Fonteyne, Margot
    et al.
    Correia, Ana
    De Plecker, Sofie
    Vercruysse, Jurgen
    Ilic, Ilija
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Veryaet, Chris
    Remon, Jean Paul
    Onofre, Fernanda
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    De Beer, Thomas
    Impact of microcrystalline cellulose material attributes: A case study on continuous twin screw granulation2015In: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 478, no 2, p. 705-717Article in journal (Refereed)
    Abstract [en]

    The International Conference on Harmonisation (ICH) states in its Q8 'Pharmaceutical Development' guideline that the manufacturer of pharmaceuticals should have an enhanced knowledge of the product performance over a range of material attributes, manufacturing process options and process parameters. The present case study evaluates the effect of unspecified variability of raw material properties upon the quality attributes of granules; produced using a continuous from-powder-to-tablet wet granulation line (ConsiGma (TM) 25). The impact of different material attributes of six samples of microcrystalline cellulose (MCC) was investigated. During a blind study the different samples of MCC were used separately and the resulting granules were evaluated in order to identify the differences between the six samples. Variation in size distribution due to varying water binding capacity of the MCC samples was observed. The cause of this different water binding capacity was investigated and was caused by a different degree of crystallinity. Afterwards, an experimental design was conducted in order to evaluate the effect of both product and process variability upon the granule size distribution. This model was used in order to calculate the required process parameters to obtain a preset granule size distribution regardless of the type of MCC used. The difference in water binding capacity and its effect on granular properties was still present when combining the MCC grades with different binders.

  • 94. Ford, Kristina L.
    et al.
    Chin, Tony
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zeng, Wei
    Doblin, Monika S.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bacic, Antony
    Comparative "Golgi" Proteome Study of Lolium multiflorum and Populus trichocarpa2016In: PROTEOMES, ISSN 2227-7382, Vol. 4, no 3, article id 23Article in journal (Refereed)
    Abstract [en]

    The Golgi apparatus (GA) is a crucial organelle in the biosynthesis of non-cellulosic polysaccharides, glycoproteins and proteoglycans that are primarily destined for secretion to the cell surface (plasma membrane, cell wall and apoplast). Only a small proportion of the proteins involved in these processes have been identified in plants, with the majority of their functions still unknown. The availability of a GA proteome would greatly assist plant biochemists, cell and molecular biologists in determining the precise function of the cell wall-related proteins. There has been some progress towards defining the GA proteome in the model plant system Arabidopsis thaliana, yet in commercially important species, such as either the cereals or woody species there has been relatively less progress. In this study, we applied discontinuous sucrose gradient centrifugation to partially enrich GA from suspension cell cultures (SCCs) and combined this with stable isotope labelling (iTRAQ) to determine protein sub-cellular locations. Results from a representative grass species, Italian ryegrass (Lolium multiflorum) and a dicot species, black cottonwood (Populus trichocarpa) are compared. The results confirm that membrane fractionation approaches that provide effective GA-enriched fractions for proteomic analyses in Arabidopsis are much less effective in the species examined here and highlight the complexity of the GA, both within and between species.

  • 95. Fortunati, E.
    et al.
    Armentano, I.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Iannoni, A.
    Saino, E.
    Visai, L.
    Berglund, Lars A.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Kenny, J. M.
    Multifunctional bionanocomposite films of poly(lactic acid), cellulose nanocrystals and silver nanoparticles2012In: Carbohydrate Polymers, ISSN 0144-8617, E-ISSN 1879-1344, Vol. 87, no 2, p. 1596-1605Article in journal (Refereed)
    Abstract [en]

    Nanocomposite films were prepared by the addition of cellulose nanocrystals (CNCs) eventually surfactant modified (s-CNC) and silver (Ag) nanoparticles in the polylactic acid (PLA) matrix using melt extrusion followed by a film formation process. Multifunctional composite materials were investigated in terms of morphological, mechanical, thermal and antibacterial response. The nanocomposite films maintained the transparency properties of the PLA matrix. Thermal analysis showed increased values of crystallinity in the nanocomposites, more evident in the s-CNC based formulations that had the highest tensile Young modulus. The presence of surfactant favoured the dispersion of cellulose nanocrystals in the polymer matrix and the nucleation effect was remarkably enhanced. Moreover, an antibacterial activity against Staphylococcus aureus and Escherichia coil cells was detected for ternary systems, suggesting that these novel nanocomposites may offer good perspectives for food packaging applications which require an antibacterial effect constant over time. (C) 2011 Elsevier Ltd. All rights reserved.

  • 96. Fortunati, E.
    et al.
    Armentano, I.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Puglia, D.
    Terenzi, A.
    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.
    Kenny, J. M.
    Microstructure and nonisothermal cold crystallization of PLA composites based on silver nanoparticles and nanocrystalline cellulose2012In: Polymer degradation and stability, ISSN 0141-3910, E-ISSN 1873-2321, Vol. 97, no 10, p. 2027-2036Article in journal (Refereed)
    Abstract [en]

    Poly(lactic acid) (PIA) based high performance nanocomposites, were prepared using an innovative combination of nanocrystalline cellulose and silver nanoparticles. Binary and ternary systems were prepared by solvent casting process and their morphological, mechanical and thermal responses were investigated. Pristine (CNC) and surfactant modified cellulose nanocrystals (s-CNC) and silver (Ag) nanoparticles were used, and the effect of cellulose crystal nano-dimension, cellulose modification, and the combination of cellulose nanostructures with silver nanoparticles, was investigated. The important industrial problem of slow crystallization of PIA was addressed by the use of cellulose nanocrystals as biobased nucleating agents and the nonisothermal cold crystallization behaviour of reinforced binary and ternary systems was studied. The presence of surfactant on the nanocrystal surface favoured the dispersion of CNC in the PLA matrix while the thermal investigations and the nonisothermal crystallization studies underlined the ability of s-CNC to act as nucleation agent in both binary and ternary nanocomposites.

  • 97.
    Fugelstad, Johanna
    KTH, School of Biotechnology (BIO), Glycoscience.
    Cellulose Biosynthesis in Oomycetes2008Licentiate thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Oomycetes have long been considered as a separate class within the kingdom Fungi, but they are in fact closer to brown algae. They are currently classified in the Stramenopile eukaryotic kingdom, which includes heterokont algae and water molds. The major cell wall polysaccharides in Oomycetes are b-(1à3) and b-(1à6)-glucans, as well as cellulose, which has never been reported in any fungal species. Chitin - the major cell wall polysaccharide in fungi - occurs in minor amounts in the walls of some Oomycetes. Some Oomycete species are pathogens of great economical importance. For example, species of the genus Phytophthora are well studied plant pathogens that cause considerable economical losses in agriculture. Saprolegniosis, a fish disease caused by species from the genus Saprolegnia, is a major problem in the aquaculture industry and represents a threat to populations of salmonids in natural habitats. Currently, there are no chemicals available that are at the same time efficient Oomycete inhibitors, environmentally friendly and safe for human consumption of treated fishes. The biosynthesis of cellulose in Oomycetes is poorly understood, even though this biochemical pathway represents a potential target for new Oomycete inhibitors. In this work, cellulose biosynthesis was investigated in two selected Oomycetes, the plant pathogen Phytophthora infestans and the fish pathogen Saprolegnia monoica.

    A new Oomycete CesA gene family was identified. It contains four homologues designated as CesA1, CesA2, CesA3 and CesA4. The gene products of CesA1, 2 and 4 contain Pleckstrin Homology domains located at the N-terminus. This represents a novel feature, unique to the Oomycete CesA genes. CesA3 is the dominantly expressed CesA homologue in the mycelium of both S. monoica and P. infestans, while CesA1 and CesA2 are up-regulated in virulent life stages of P. infestans. CesA4 was expressed only in minute amounts in all investigated types of cells. Gene silencing by RNA interference of the whole CesA gene family in P. infestans lead to decreased amounts of cellulose in the cell wall. The inhibitors of cellulose synthesis DCB and Congo Red had an up-regulating effect on SmCesA gene expression, which was accompanied by an increased b-glucan synthase activity in vitro. In addition, these inhibitors slowed down the growth of the mycelium from S. monoica. Zoospores from P. infestans treated with DCB were unable to infect potato leaves and showed aberrant cell wall morphologies similar to those obtained by silencing the CesA gene family.

    Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that the synthesis of cellulose is crucial for infection of potato by P. infestans

  • 98.
    Fugelstad, Johanna
    KTH, School of Biotechnology (BIO), Glycoscience.
    Functional characterization of cellulose and chitin synthase genes in Oomycetes2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Some species of Oomycetes are well studied pathogens that cause considerable economical losses in the agriculture and aquaculture industries. Currently, there are no chemicals available that are environmentally friendly and at the same time efficient Oomycete inhibitors. The cell wall of Oomycetes consists of b-(1à3) and b-(1à6)-glucans, cellulose and in some species minute amounts of chitin. The biosynthesis of cellulose and chitin in Oomycetes is poorly understood. However, cell wall synthesis represents a potential target for new Oomycete inhibitors. In this work, cellulose and chitin synthase genes and gene products were analyzed in the plant pathogen Phytophthora infestans and in the fish pathogen Saprolegnia monoica.

     

    A new Oomycete CesA gene family was identified, containing four subclasses of genes designated as CesA1 to 4. The gene products of CesA1, 2 and 4 contain pleckstrin homology (PH) domains located at the N-terminus, which is unique to the Oomycete CesAs. Our results show that the SmCesA2 PH domain binds to phosphoinositides, F-actin and microtubules in vitro and can co-localize with F-actin in vivo. Functional characterization of the CesA genes by gene silencing in P. infestans led to decreased cellulose content in the cell wall. The cellulose synthase inhibitors DCB and Congo Red inhibited the growth of the mycelium of S. monoica and had an up-regulating effect on SmCesA gene expression. Zoospores from P. infestans treated with DCB were unable to infect potato leaves. In addition, two full-length chitin synthase genes (Chs) were analyzed from S. monoica.  Expression of SmChs2 in yeast yielded an active recombinant protein. The biochemical characterization of the in vitro product of SmChs2 confirmed that the protein is responsible for chitin formation. The chitin synthase inhibitor nikkomycin Z inhibited the SmChs2 both in vivo and in vitro.

     

    Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that synthesis of cellulose is crucial for infection of potato by P. infestans. The PH domain is involved in the interaction of CesA with the cytoskeleton. In addition, we firmly demonstrate that the SmChs2 gene encodes a catalytically active chitin synthase.

  • 99.
    Fugelstad, Johanna
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bouzenzana, Jamel
    Djerbi, Soraya
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Numerical Analysis and Computer Science, NADA.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    A novel family of cellulose synthase genes from the Oomycete Saprolegnia monoica: functional characterization using cellulose synthesis inhibitorsManuscript (Other academic)
  • 100.
    Fugelstad, Johanna
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bouzenzana, Jamel
    Djerbi, Soraya
    Guerriero, Gea
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Identification of the cellulose synthase genes from the Oomycete Saprolegnia monoica and effect of cellulose synthesis inhibitors on gene expression and enzyme activity2009In: Fungal Genetics and Biology, ISSN 1087-1845, E-ISSN 1096-0937, Vol. 46, no 10, p. 759-767Article in journal (Refereed)
    Abstract [en]

    Cellulose biosynthesis is a vital but yet poorly understood biochemical process in Oomycetes. Here, we report the identification and characterization of the cellulose synthase genes (CesA) from Saprolegnia monoica. Southern blot experiments revealed the occurrence of three CesA homologues in this species and phylogenetic analyses confirmed that Oomycete CesAs form a clade of their own. All gene products contained the D,D,D,QXXRW signature of most processive glycosyltransferases, including cellulose synthases. However, their N-terminal ends exhibited Oomycete-specific domains, i.e. Pleckstrin Homology domains, or conserved domains of an unknown function together with additional putative transmembrane domains. Mycelial growth was inhibited in the presence of the cellulose biosynthesis inhibitors 2,6-dichlorobenzonitrile or Congo Red. This inhibition was accompanied by a higher expression of all CesA genes in the mycelium and increased in vitro glucan synthase activities. Altogether, our data strongly suggest a direct involvement of the identified CesA genes in cellulose biosynthesis.

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