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  • 251.
    Porsch, Christian
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Zhang, Yuning
    Ducani, Cosimo
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Nordstierna, Lars
    Nyström, Andreas M.
    Malmström, Eva
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Toward Unimolecular Micelles with Tunable Dimensions Using Hyperbranched Dendritic-Linear Polymers2014In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 15, no 6, p. 2235-2245Article in journal (Refereed)
    Abstract [en]

    A library of amphiphilic, hyperbranched dendritic-linear polymers (HBDLPs) are successfully synthesized, and evaluated as potential unimolecular micelles. Hyperbranched macroinitiators (HBMI), extended with poly(ethylene glycol) methacrylate (P(OEGMA)), are afforded via a combination of self-condensing vinyl (co)polymerization (SCV(C)P) and atom transfer radical polymerization (ATRP), providing a versatile two-step synthetic route. The HBDLP architecture and chain lengths are varied, and the effect on the nanoparticle (NP) stability and properties are evaluated. The HBDLPs form predominantly stable and spherical NPs, and the NP dimensions could be tailored by the HBDLP characteristics. The NPs formed are of high molecular weight, and their stability varies with the properties of the corresponding HBDLP. Too small dendritic segment, or too low degree of PEGylation, results to some extent in NP aggregation, while higher molecular weight HBDLPs, with a high amount of hydrophilic segments, appears to form discrete unimolecular micelles. The versatility of the platform is further demonstrated by the convenience of forming a HBDLP. with a more complex, linear copolymer extension instead of P(OEGMA).

  • 252.
    Prakobna, Kasinee
    et al.
    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.
    Terenzi, Camilla
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. 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.
    Furo, Istvan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. KTH, School of Chemical Science and Engineering (CHE), Centres, Industrial NMR Centre.
    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.
    Core-shell cellulose nanofibers for biocomposites: Nanostructural effects in hydrated state2015In: Carbohydrate Polymers, ISSN 0144-8617, E-ISSN 1879-1344, Vol. 125, p. 92-102Article in journal (Refereed)
    Abstract [en]

    Core-shell wood cellulose nanofibers (CNF) coated by an XG hemicellulose polymer are prepared and used to make biocomposites. CNF/XG biocomposites have interest as packaging materials and as hydrated CNF/XG plant cell wall analogues. Structure and properties are compared between Core-shell CNF/XG and more inhomogeneous CNF/XG. Experiments include XG sorption, dynamic light scattering of CNF nanoparticle suspensions, FE-SEM of nanostructure, moisture sorption, tensile testing in moist conditions and dynamic mechanical analysis. (2)H NMR relaxometry is performed on materials containing sorbed (2)H2O2 in order to assess water molecular dynamics in different materials. The results clarify the roles of CNF, XG and the CNF/XG interface in the biocomposites, both in terms of moisture sorption mechanisms and mechanical properties in moist state. The concept of core-shell nanofiber network biocomposites, prepared by filtering of colloids, provides improved control of polymer matrix distribution and interface structure. Also, present mechanical properties are much superior to comparable plant fiber biocomposites.

  • 253. Purushotham, P.
    et al.
    Cho, S. H.
    Díaz-Moreno, Sara M
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kumar, M.
    Nixon, B. T.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, Australia.
    Zimmer, J.
    A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 40, p. 11360-11365Article in journal (Refereed)
    Abstract [en]

    Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme's N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.

  • 254.
    Rajangam, Alex S.
    et al.
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Kumar, Manoj
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Guerriero, Gea
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Pansri, Podjamas
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Brown, Christian J. L.
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Blomqvist, Kristina
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Mellerowicz, Ewa
    Sundberg, Bjorn
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    MAP20, a Microtubule-Associated Protein in the Secondary Cell Walls of Hybrid Aspen, Is a Target of the Cellulose Synthesis Inhibitor 2,6-Dichlorobenzonitrile2008In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 148, no 3, p. 1283-1294Article in journal (Refereed)
    Abstract [en]

    We have identified a gene, denoted PttMAP20, which is strongly up-regulated during secondary cell wall synthesis and tightly coregulated with the secondary wall-associated CESA genes in hybrid aspen (Populus tremula x tremuloides). Immunolocalization studies with affinity-purified antibodies specific for PttMAP20 revealed that the protein is found in all cell types in developing xylem and that it is most abundant in cells forming secondary cell walls. This PttMAP20 protein sequence contains a highly conserved TPX2 domain first identified in a microtubule-associated protein (MAP) in Xenopus laevis. Overexpression of PttMAP20 in Arabidopsis (Arabidopsis thaliana) leads to helical twisting of epidermal cells, frequently associated with MAPs. In addition, a PttMAP20-yellow fluorescent protein fusion protein expressed in tobacco (Nicotiana tabacum) leaves localizes to microtubules in leaf epidermal pavement cells. Recombinant PttMAP20 expressed in Escherichia coli also binds specifically to in vitro-assembled, taxol-stabilized bovine microtubules. Finally, the herbicide 2,6-dichlorobenzonitrile, which inhibits cellulose synthesis in plants, was found to bind specifically to PttMAP20. Together with the known function of cortical microtubules in orienting cellulose microfibrils, these observations suggest that PttMAP20 has a role in cellulose biosynthesis.

  • 255. Robles, E.
    et al.
    Salaberria, A. M.
    Herrera, R.
    Fernandes, Susana C. M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Labidi, J.
    Self-bonded composite films based on cellulose nanofibers and chitin nanocrystals as antifungal materials2016In: Carbohydrate Polymers, ISSN 0144-8617, E-ISSN 1879-1344, Vol. 144, p. 41-49Article in journal (Refereed)
    Abstract [en]

    Cellulose nanofibers and chitin nanocrystals, two main components of agricultural and aquacultural by-products, were obtained from blue agave and yellow squat lobster industrial residues. Cellulose nanofibers were obtained using high pressure homogenization, while chitin nanocrystals were obtained by hydrolysis in acid medium. Cellulose nanofibers and chitin nanocrystals were characterized by X-ray diffraction, Atomic Force Microscopy and Infrared spectroscopy. Self-bonded composite films with different composition were fabricated by hot pressing and their properties were evaluated. Antifungal activity of chitin nanocrystals was studied using a Cellometer®cell count device, mechanical properties at tension were measured with a universal testing machine, water vapor permeability was evaluated with a thermohygrometer and surface tension with sessile drop contact angle method. The addition of chitin nanocrystals reduced slightly the mechanical properties of the composite. Presence of chitin nanocrystals influenced the growth of Aspergillus sp fungus in the surface of the composites as expected.

  • 256. Rueda, L.
    et al.
    Fernandez d'Arlas, B.
    Zhou, Qi
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. 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.
    Corcuera, M. A.
    Mondragon, I.
    Eceiza, A.
    Isocyanate-rich cellulose nanocrystals and their selective insertion in elastomeric polyurethane2011In: Composites Science And Technology, ISSN 0266-3538, E-ISSN 1879-1050, Vol. 71, no 16, p. 1953-1960Article in journal (Refereed)
    Abstract [en]

    Cellulose nanocrystals (CNC) were successfully obtained and modified with 1,6-hexamethylene diisocyanate (HE)!) by means of in situ polymerization varying the CNC/HDI molar ratio to evaluate the number of anchored chains to the CNC. The modification was examined by elemental analysis, nuclear magnetic resonance ((13)C NMR) and attenuated total reflection Fourier transform infrared spectroscopy (IR-ATR). Nanocomposites containing 1.5 wt% CNC, modified and unmodified, were prepared by solvent casting. Thermal and mechanical properties of the resulting films were evaluated from the viewpoint of polyurethane microphase separated structure, soft and hard domains. CNC were effectively dispersed in the polyurethane matrix and depending on surface chemistry, the nanoreinforcement interacts selectively with matrix nanodomains. This interpretation is supported by differences in thermal and mechanical properties of the nanocomposites and also confirmed by AFM images. Isocyanate rich cellulose nanocrystals interacted with matrix hard phase, promoting physical association with hard segments, enhancing stiffness and dimensional stability versus temperature of the nanocomposite.

  • 257. Rueda, L.
    et al.
    Saralegui, A.
    Fernandez d'Arlas, B.
    Zhou, Qi
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Biotechnology (BIO), Glycoscience.
    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.
    Corcuera, M. A.
    Mondragon, I.
    Eceiza, A.
    Cellulose nanocrystals/polyurethane nanocomposites. Study from the viewpoint of microphase separated structure2013In: Carbohydrate Polymers, ISSN 0144-8617, E-ISSN 1879-1344, Vol. 92, no 1, p. 751-757Article in journal (Refereed)
    Abstract [en]

    Cellulose nanocrystals (CNC) successfully obtained from microcrystalline cellulose (MCC) were dispersed in a thermoplastic polyurethane as matrix. Nanocomposites containing 1.5.5. 10 and 30 wt% CNC were prepared by solvent casting procedure and properties of the resulting films were evaluated from the viewpoint of polyurethane microphase separated structure, soft and hard domains. CNC were effectively dispersed in the segmented thermoplastic elastomeric polyurethane (STPUE) matrix due to the favorable matrix-nanocrystals interactions through hydrogen bonding. Cellulose nanocrystals interacted with both soft and hard segments, enhancing stiffness and stability versus temperature of the nanocomposites. Thermal and mechanical properties of STPUE/CNC nanocomposites have been associated to the generated morphologies investigated by AFM images.

  • 258. Ruprecht, Colin
    et al.
    Tohge, Takayuki
    Fernie, Alisdair
    Mortimer, Cara L.
    Kozlo, Amanda
    Fraser, Paul D.
    Funke, Norma
    Cesarino, Igor
    Vanholme, Ruben
    Boerjan, Wout
    Morreel, Kris
    Burgert, Ingo
    Gierlinger, Notburga
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Schneider, Vera
    Stockero, Andrea
    Navarro, Juan Pedro
    Pudel, Frank
    Tambuyser, Bart
    Hygate, James
    Bumstead, Jon
    Notley, Louis
    Persson, Staffan
    Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry2014In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 87, p. e51393-Article in journal (Refereed)
    Abstract [en]

    The global demand for food, feed, energy and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

  • 259.
    Ruthes, Andrea C.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Martinez-Abad, Antonio
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tan, Hwei-Ting
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, Australia.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, Australia.
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience.
    Sequential fractionation of feruloylated hemicelluloses and oligosaccharides from wheat bran using subcritical water and xylanolytic enzymes2017In: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 19, no 8, p. 1919-1931Article in journal (Refereed)
    Abstract [en]

    Wheat bran is a major by-product of cereal production that still has limited use for advanced nutritional and material applications. A sequential process using subcritical water, membrane filtration and selective enzymatic treatments has been designed for the combined fractionation of functional high molar mass hemicelluloses (over 10(5) g mol(-1)) and oligosaccharides from wheat bran. This process not only offers increased total solid yield compared with conventional protocols based on alkaline extraction, but it also preserves the inherent functionalities of the phenolic groups that substitute the carbohydrate structures of the extracted hemicelluloses. Feruloylated arabinoxylans (F-AX) with high molar mass and significant radical scavenging activity can be isolated from the subcritical water extract. Structurally different oligosaccharides, including mixed-linkage beta-D-glucan oligosaccharides (BGOs) and arabinoxylo-oligosaccharides (AXOs) can be recovered from the eluent after membrane filtration. The crosslinked residue after subcritical water extraction was further treated with xylanolytic enzymes to release valuable feruloylated arabinoxylo-oligosaccharides (FAXOs). The oligo-and polysaccharide fractions isolated from this sequential process show great potential for use as prebiotic or platform chemicals, and as polymeric matrices for carbohydrate-based materials with radical scavenging properties, respectively.

  • 260.
    Ruthes, Andrea C.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Smiderle, Fhernanda R.
    Iacomini, Marcello
    Mushroom heteropolysaccharides: A review on their sources, structure and biological effects2016In: Carbohydrate Polymers, ISSN 0144-8617, E-ISSN 1879-1344, Vol. 136, p. 358-375Article, review/survey (Refereed)
    Abstract [en]

    Mushrooms have been largely studied not only by their D-glucans, but also because they present a class of more complex polymers: the heteropolysaccharides. Heteropolysaccharides show variability on their monosaccharide composition, anomeric configuration, linkage and branching type, besides some of these molecules can present natural methylated monosaccharides and also acid monosaccharides, which enhance the difficulty of the purification and characterization of their structure. As a result of such complexity, mushroom heteropolysaccharides can be considered an interesting source of molecules with medicinal and industrial applications. Consequently a plenty of new researches has been published in the past 12 years about the isolation, chemical characterization and biological activities of heteropolysaccharides from mushrooms, especially from Basidiomycetes. Therefore, this review intends to organize and classify the information described up to now about such polysaccharides obtained from different sources of mushroom-forming fungi, Basidiomycetes, Ascomycetes and Hybrid mushrooms, and provides a brief reflection on how the chemical studies have been carried out.

  • 261. Salaberria, Asier M.
    et al.
    Labidi, Jalel
    Fernandes, Susana C. M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Different routes to turn chitin into stunning nano-objects2015In: European Polymer Journal, ISSN 0014-3057, E-ISSN 1873-1945, Vol. 68, p. 503-515Article in journal (Refereed)
    Abstract [en]

    Due to its intractable structure and inherent insoluble nature, chitin was for a long time an underutilized resource. The increasing interest in the use of chitin as a source of nanostructured materials is quite recent. This review provides the latest advances in different ways to isolate or fabricate chitin nano-objects - chitin nanocrystals (CHNC) and chitin nanofibers (CHNF) - from different chitin sources. It also summarizes the chronology of some important scientific advances on chitin research during its 200 years of history. Additionally, engineered composite materials based on chitin nano-objects are reviewed.

  • 262. Salaheddin, Clara
    et al.
    Spadiut, Oliver
    Ludwig, Roland
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Glycoscience.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Haltrich, Dietmar
    Peterbauer, Clemens
    Probing active-site residues of pyranose 2-oxidase from Trametes multicolor by semi-rational protein design.2009In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 4, no 4, p. 535-543Article in journal (Refereed)
    Abstract [en]

    D-Tagatose is a sweetener with low caloric and non-glycemic characteristics. It can be produced by an enzymatic oxidation of D-galactose specifically at C2 followed by chemical hydrogenation. Pyranose 2-oxidase (P2Ox) from Trametes multicolor catalyzes the oxidation of many aldopyranoses to their corresponding 2-keto derivatives. Since D-galactose is not the preferred substrate of P2Ox, semi-rational design was employed to improve the catalytic efficiency with this poor substrate. Saturation mutagenesis was applied on all positions in the active site of the enzyme, resulting in a library of mutants, which were screened for improved activity in a 96-well microtiter plate format. Mutants with higher activity than wild-type P2Ox were chosen for further kinetic investigations. Variant V546C was found to show a 2.5-fold increase of k(cat) with both D-glucose and D-galactose when oxygen was used as electron acceptor. Because of weak substrate binding, however, k(cat)/K(M) is lower for both sugar substrates compared to wild-type TmP2Ox. Furthermore, variants at position T169, i.e., T169S and T169N, showed an improvement of the catalytic characteristics of P2Ox with D-galactose. Batch conversion experiments of D-galactose to 2-keto-D-galactose were performed with wild-type TmP2O as well as with variants T169S, T169N, V546C and V546C/T169N to corroborate the kinetic properties determined by Michaelis-Menten kinetics.

  • 263.
    Salajkova, Michaela
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. Brno University of Technology, Czech Republic .
    Sehaqui, Houssine
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Zhou, Qi
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Biotechnology (BIO), Glycoscience.
    Berglund, Lars A.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Nanostructured composite materials from microfibrillated cellulose and carbon nanotubes2009In: ICCM-17 17th International Conference on Composite Materials, 2009Conference paper (Refereed)
    Abstract [en]

    Thin composite films were prepared from the mixture of the aqueous suspension of microfibrillated cellulose (MFC) and multi-walled carbon nanotubes (MWCNTs). The morphology, electrical conductivity, and mechanical properties of the composites were characterized. Good electrical properties were obtained when the MWCNTs content was higher than 2 wt%.

  • 264.
    Salajkova, Michaela
    et al.
    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.
    Valentini, Luca
    Zhou, Qi
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites. KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Berglund, Lars
    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.
    Tough nanopaper structures based on cellulose nanofibers and carbon nanotubes2013In: Composites Science And Technology, ISSN 0266-3538, E-ISSN 1879-1050, Vol. 87, p. 103-110Article in journal (Refereed)
    Abstract [en]

    Carbon nanotube (CNT) nanocomposites based on CNT in a polymer matrix typically have low strain to failure in tensile loading. Furthermore, mixing of more than a few percent of CNT with either molten thermoplastics or monomers in bulk often results in agglomeration of CNT. Here, multiwalled CNT (MWCNT) are mixed with nanofibrillated cellulose (NFC) in aqueous suspension and filtered into tough nanopaper structures with up to 17 wt% of MWCNT commingled with NFC nanofibrils. Carbon nanotubes were surface treated with a surfactant, and homogenous suspensions of carbon nanotubes in water miscible with the NFC suspension was obtained. NFC/CNT nanopaper structures were characterized for porosity using mercury displacement, and studied by FE-SEM and AFM. Mechanical properties were tested in uniaxial tension and electrical conductivity was measured. The processing route is environmentally friendly and leads to well-mixed structures. Thin coatings as well as thicker films can be prepared, which show a combination of high electrical conductivity, flexibility in bending and high tensile strength.

  • 265. Samalova, Marketa
    et al.
    Melida, Hugo
    KTH, School of Biotechnology (BIO), Glycoscience.
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Soanes, Darren M.
    Talbot, Nicholas J.
    Gurr, Sarah J.
    The beta-1,3-glucanosyltransferases (Gels) affect the structure of the rice blast fungal cell wall during appressorium-mediated plant infection2017In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 19, no 3, article id UNSP e12659Article in journal (Refereed)
    Abstract [en]

    The fungal wall is pivotal for cell shape and function, and in interfacial protection during host infection and environmental challenge. Here, we provide the first description of the carbohydrate composition and structure of the cell wall of the rice blast fungus Magnaporthe oryzae. We focus on the family of glucan elongation proteins (Gels) and characterize five putative beta-1,3-glucan glucanosyltransferases that each carry the Glycoside Hydrolase 72 signature. We generated targeted deletion mutants of all Gel isoforms, that is, the GH72(+), which carry a putative carbohydrate-binding module, and the GH72(-)Gels, without this motif. We reveal that M. oryzae GH72(+) GELs are expressed in spores and during both infective and vegetative growth, but each individual Gel enzymes are dispensable for pathogenicity. Further, we demonstrated that Delta gel1 Delta gel3 Delta gel4 null mutant has a modified cell wall in which 1,3-glucans have a higher degree of polymerization and are less branched than the wild-type strain. The mutant showed significant differences in global patterns of gene expression, a hyper-branching phenotype and no sporulation, and thus was unable to cause rice blast lesions (except via wounded tissues). We conclude that Gel proteins play significant roles in structural modification of the fungal cell wall during appressorium-mediated plant infection.

  • 266. Sandh, Gustaf
    et al.
    Ran, Liang
    Xu, Linghua
    Sundqvist, Gustav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bergman, Birgitta
    Comparative proteomic profiles of the marine cyanobacterium Trichodesmium erythraeum IMS101 under different nitrogen regimes2011In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 11, no 3, p. 406-419Article in journal (Refereed)
    Abstract [en]

    Trichodesmium is a marine filamentous diazotrophic cyanobacterium and an important contributor of "new" nitrogen in the oligotrophic surface waters of the tropical and subtropical oceans. It is unique in that it exclusively fixes N-2 at daytime, although it belongs to the non-heterocystous filamentous segment of the cyanobacterial radiation. Here we present the first quantitative proteomic analysis of Trichodesmium erythraeum IMS101 when grown under different nitrogen regimes using 2-DE/MALDI-TOF-MS. Addition of combined nitrogen (NO3-) prevented development of the morphological characteristics of the N-2-fixing cell type (diazocytes), inhibited expression of the nitrogenase enzyme subunits and consequently N-2 fixation activity. The diazotrophic regime (N-2 versus NO3- cultures) elicited the differential expression of more than 100 proteins, which represented 13.5% of the separated proteins. Besides proteins directly related to N-2 fixation, proteins involved in the synthesis of reducing equivalents and the generation of a micro-oxic environment were strongly up-regulated, as was in particular Dps, a protein related to iron acquisition and potentially other vital cellular processes. In contrast, proteins involved in the S-adenosylmethionine (SAM) cycle, synthesis of amino acids and production of carbon skeletons for storage and synthesis of amino acids were suppressed. The data are discussed in the context of Trichodesmium's unusual N-2-fixing physiology.

  • 267. Saraiva, Marcia
    et al.
    De Bruijn, Irene
    Grenville-Briggs, Laura
    KTH, School of Biotechnology (BIO), Glycoscience. Aberdeen Oomycete Laboratory, University of Aberdeen, United Kingdom; Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Sweden.
    Mclaggan, Debbie
    Willems, Ariane
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Van West, Pieter
    Functional characterization of a tyrosinase gene from the oomycete Saprolegnia parasitica by RNAi silencing2014In: Fungal Biology, ISSN 1878-6146, E-ISSN 1878-6162, Vol. 118, no 7, p. 621-629Article in journal (Refereed)
    Abstract [en]

    Here we describe the first application of transient gene silencing in Saprolegnia parasitica, a pathogenic oomycete that infects a wide range of fish, amphibians, and crustaceans. A gene encoding a putative tyrosinase from S. parasitica, SpTyr, was selected to investigate the suitability of RNA-interference (RNAi) to functionally characterize genes of this economically important pathogen. Tyrosinase is a mono-oxygenase enzyme that catalyses the O-hydroxylation of monophenols and subsequent oxidation of O-diphenols to quinines. These enzymes are widely distributed in nature, and are involved in the melanin biosynthesis. Gene silencing was obtained by delivering in vitro synthesized SpTyr dsRNA into protoplasts. Expression analysis, tyrosinase activity measurements, and melanin content analysis confirmed silencing in individual lines. Silencing of SpTyr resulted in a decrease of tyrosinase activity between 38 % and 60 %, dependent on the level of SpTyr-expression achieved. The SpTyr-silenced lines displayed less pigmentation in developing sporangia and occasionally an altered morphology. Moreover, developing sporangia from individual silenced lines possessed a less electron dense cell wall when compared to control lines, treated with GFP-dsRNA. In conclusion, the tyrosinase gene of S. parasitica is required for melanin formation and transient gene silencing can be used to functionally characterize gene S in S. parasitica.

  • 268. Saura-Valls, M.
    et al.
    Faure, R.
    Ragas, S.
    Piens, K.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Cottaz, S.
    Driguez, H.
    Planas, A.
    Kinetic analysis using low-molecular mass xyloglucan oligosaccharides defines the catalytic mechanism of a Populus xyloglucan endotransglycosylase2006In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 395, p. 99-106Article in journal (Refereed)
    Abstract [en]

    Plant XETs [XG (xyloglucan) endotransglycosylases] catalyse the transglycosylation front a XG donor to a XG or low-molecular-mass XG fragment Lis the acceptor, and are thought to be important enzymes in the formation and remodelling of the cellulose-XG three-dimensional network in the primary plant cell wall. Current methods to assay XET activity use the XG polysaccharide as the donor substrate, and present limitations for kinetic and mechanistic studies of XET action due to the polymeric and polydisperse nature of the substrate. A novel activity assay based oil HPCE (high performance capillary electrophoresis), in con, junction with a defined low-molecular-mass XGO {XG oligosaccharicle; (XXXGXXXG, where G = Glc beta 1,4- and X = [Xyl alpha 1,6]Glc beta 1,4-)l as the glycosyl donor and a heptasaccharide derivatized with ANTS [8-aminonaphthalene-1,3,6-trisulphonic acid; (XXXG-ANTS)] as the acceptor substrate was developed and validated. The recombinant enzyme PttXET16A from Popidus tremula x tremuloides (hybrid aspen) was characterized using file donor/acceptor pair indicated above, for which preparative scale syntheses have been optimized. The low-molecular-mass donor underwent a single transglycosylation reaction to the acceptor substrate under initial-rate conditions. with a pH optimum at 5.0 and maximal activity between 30 and 40 degrees C. Kinetic data are best explained by a ping-pong bi-bi mechanism With Substrate inhibition by both donor and acceptor. This is the first assay for XETs using a donor Substrate other than polymeric XG, enabling quantitative kinetic analysis of different XGO donors for specificity, and subsite mapping studies of XET enzymes.

  • 269. Saura-Valls, Marc
    et al.
    Faure, Regis
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Cottaz, Sylvain
    Driguez, Hugues
    Planas, Antoni
    Active-site mapping of a Populus xyloglucan endo-transglycosylase with a library of xylogluco-oligosaccharides2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 32, p. 21853-21863Article in journal (Refereed)
    Abstract [en]

    Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16-34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites -1 and + 1). Donor binding is dominated by the higher affinity of the XXXGmoiety (G = Glc beta(1 -> 4) and X = Xyl alpha(1 -> 6)Glc beta(1 -> 4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with k(cat)/K-m values for the donor substrates showed that a GG-unit on negative and-XXG on positive subsites are the minimal requirements for activity. Subsites -2 and -3 (for backbone Glc residues) and +2' (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal = Gal beta(1 -> 4)) is the best donor substrate with a "blocked" nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.

  • 270. Schrick, Kathrin
    et al.
    DeBolt, Seth
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Deciphering the molecular functions of sterols in cellulose biosynthesis2012In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 3, p. 84-Article in journal (Refereed)
    Abstract [en]

    Sterols play vital roles in plant growth and development, as components of membranes and as precursors to steroid hormones. Analysis of Arabidopsis mutants indicates that sterol composition is crucial for cellulose biosynthesis. Sterols are widespread in the plasma membrane (PM), suggesting a possible link between sterols and the multimeric cellulose synthase complex. In one possible scenario, molecular interactions in sterol-rich PM microdomains or another form of sterol-dependent membrane scaffolding may be critical for maintaining the correct subcellular localization, structural integrity and/or activity of the cellulose synthase machinery. Another possible link may be through steryl glucosides, which could act as primers for the attachment of glucose monomers during the synthesis of beta - (1 -> 4) glucan chains that form the cellulose microfibrils. This mini-review examines genetic and biochemical data supporting the link between sterols and cellulose biosynthesis in cell wall formation and explores potential approaches to elucidate the mechanism of this association.

  • 271.
    Sehaqui, Houssine
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Liu, Andong
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Berglund, Lars A.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Fast Preparation Procedure for Large, Flat Cellulose and Cellulose/Inorganic Nanopaper Structures2010In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 11, no 9, p. 2195-2198Article in journal (Refereed)
    Abstract [en]

    Nanostructured materials are difficult to prepare rapidly and as large structures. The present study is thus significant because a rapid preparation procedure for large, flat, smooth, and optically transparent cellulose nanopaper structures is developed using a semiautomatic sheet former. Cellulose/inorganic hybrid nanopaper is also produced. The preparation procedure is compared with other approaches, and the nanopaper structures are tested in uniaxial tensile tests. Optical transparency and high tensile strength are demonstrated in 200 mm diameter nanopaper sheets, indicating well-dispersed nanofibrils. The preparation time is 1 h for a typical nanopaper thickness of 60 pm. In addition, the application of the nanopaper-making strategy to cellulose/inorganic hybrids demonstrates the potential for "green" processing of new types of nanostructured functional materials.

  • 272.
    Sehaqui, Houssine
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Salajkova, Michaela
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. Brno University of Technology, Czech Republic .
    Zhou, Qi
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Biotechnology (BIO), Glycoscience.
    Berglund, Lars A.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Biomimetic aerogels from microfibrillated cellulose and xyloglucan2009In: ICCM-17 17th International Conference on Composite Materials, 2009Conference paper (Refereed)
    Abstract [en]

    Cellulose aerogels with density of 7-100 kg/m3 were prepared by freeze drying from microfibrillated cellulose water suspensions, and biomimetic aerogels were prepared with the addition of xyloglucan. Their microstructures and physical properties were characterized by scanning electron microscopy, nitrogen adsorption measurements, and tensile tests.

  • 273.
    Sehaqui, Houssine
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Salajkova, Michaela
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Zhou, Qi
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites. 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.
    Mechanical performance tailoring of tough ultra-high porosity foams prepared from cellulose I nanofiber suspensions2010In: Soft Matter, ISSN 1744-683X, Vol. 6, no 8, p. 1824-1832Article in journal (Refereed)
    Abstract [en]

    Low-density structures of mechanical function in plants, arthropods and other organisms, are often based on high- strength cellulose or chitin nanofibers and show an interesting combination of flexibility and toughness. Here, a series of plant-inspired tough and mechanically very robust cellular biopolymer foams with porosities as high as 99.5% (porosity range 93.1-99.5%) were therefore prepared by solvent-free freeze-drying from cellulose I wood nanofiber water suspensions. A wide range of mechanical properties was obtained by controlling density and nanofiber interaction in the foams, and density property relationships were modeled and compared with those for inorganic aerogels. Inspired by cellulose-xyloglucan (XG) interaction in plant cell walls, XG was added during preparation of the toughest foams. For the cellulose-XG nanocomposite foams in particular, the mechanical properties at comparable densities were superior to those reported in the literature for clay aerogel/cellulose whisker nanocomposites, epoxy/clay aerogels, polymer/clay/nanotube aerogels, and polymer/silica aerogels. The foam structure was characterized by high-resolution field-emission scanning electron microscopy and the specific surface area was measured by nitrogen physisorption. Dynamic mechanical thermal analysis and uniaxial compression tests were performed. The foam was thermally stable up to 275 degrees C where cellulose started to degrade.

  • 274.
    Sehaqui, Houssine
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Berglund, Lars A.
    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, Biocomposites.
    High-porosity aerogels of high specific surface area prepared from nanofibrillated cellulose (NFC)2011In: Composites Science And Technology, ISSN 0266-3538, E-ISSN 1879-1050, Vol. 71, no 13, p. 1593-1599Article in journal (Refereed)
    Abstract [en]

    Low-density aerogels based on nanofibrillated cellulose (NFC) from wood pulp were prepared from NFC aqueous dispersions using solvent exchange from water to tert-butanol followed by tert-butanol freeze-drying. In the present study, the dispersion of NFC nanofibers in the hydrocolloid was very well preserved in the aerogels. The "effective" diameter of the NFC nanofibers in the aerogels is around 10-18 nm corresponding to specific surface areas as high as 153-284 m(2) g(-1). Aerogels based on different NFC nanofibers were studied by FE-SEM, BET analysis (nitrogen gas adsorption), and mechanical properties were measured in compression for different densities of aerogels. The properties are compared with polymer foams and inorganic aerogels. Compared with cellular NFC foams, the present nanofibrous aerogels have lower modulus and show lower stress in compression for a given strain. Tert-butanol freeze-drying can therefore be used to create "soft" aerogels.

  • 275.
    Sehaqui, Houssine
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Zhou, Qi
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. 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.
    Nanostructured biocomposites of high toughness-a wood cellulose nanofiber network in ductile hydroxyethylcellulose matrix2011In: Soft Matter, ISSN 1744-683X, E-ISSN 1744-6848, Vol. 7, no 16, p. 7342-7350Article in journal (Refereed)
    Abstract [en]

    Nanopaper from wood-based nanofibrillated cellulose (NFC) offers vastly improved strength and strain-to-failure compared with plant fiber-based paper and plant fiber biocomposites. In the present study, unique nanostructural toughening effects are reported in cellulose nanofiber/hydroxyethylcellulose (HEC) biocomposites. HEC is an amorphous cellulose derivative of high molar mass and toughness. A previously developed preparation route inspired by paper-making is used. It is "green", scalable, and allows high reinforcement content. In the present concept, nanostructural control of polymer matrix distribution is exercised as the polymer associates with the reinforcement. This results in nanocomposites of a soft HEC matrix surrounding nanofibrillated cellulose forming a laminated structure at the submicron scale, as observed by FE-SEM. We study the effect of NFC volume fraction on tensile properties, thermomechanical stability, creep properties and moisture sorption of the nanocomposites. The results show strong property improvements with NFC content due to the load-carrying ability of the NFC network. At an NFC volume fraction of 45%, the toughness was more than doubled compared with cellulose nanopaper. The present nanocomposite is located in previously unoccupied space in a strength versus strain-to-failure property chart, outside the regions occupied by microscale composites and engineering polymers. The results emphasize the potential for extended composites mechanical property range offered by nanostructured biocomposites based on high volume fraction nanofiber networks.

  • 276.
    Sehaqui, Houssine
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Zhou, Qi
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. KTH, School of Biotechnology (BIO), Glycoscience.
    Ikkala, Olli
    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.
    Strong and Tough Cellulose Nanopaper with High Specific Surface Area and Porosity2011In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 12, no 10, p. 3638-3644Article in journal (Refereed)
    Abstract [en]

    In order to better understand nanostructured fiber networks, effects from high specific surface area of nanofibers are important to explore. For cellulose networks, this has so far only been achieved in nonfibrous regenerated cellulose aerogels. Here, nanofibrillated cellulose (NFC) is used to prepare high surface area nanopaper structures, and the mechanical properties are measured in tensile tests. The water in NFC hydrogels is exchanged to liquid CO(2), supercritical CO(2), and tert-butanol, followed by evaporation, supercritical drying, and sublimation, respectively. The porosity range is 40-86%. The nanofiber network structure in nanopaper is characterized by FE-SEM and nitrogen adsorption, and specific surface area is determined. High-porosity TEMPO-oxidized NFC nanopaper (56% porosity) prepared by critical point drying has a specific surface area as high as 48(2) m(2) g(-1). The mechanical properties of this nanopaper structure are better than for many thermoplastics, but at a significantly lower density of only 640 kg m(-3). The modulus is 1.4 GPa, tensile strength 84 MPa, and strain-to-failure 17%. Compared with water-dried nanopaper, the material is softer with substantially different deformation behavior.

  • 277. Silipo, Alba
    et al.
    Larsbrink, Johan
    KTH, School of Biotechnology (BIO), Glycoscience.
    Marchetti, Roberta
    Lanzetta, Rosa
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Molinaro, Antonio
    NMR Spectroscopic Analysis Reveals Extensive Binding Interactions of Complex Xyloglucan Oligosaccharides with the Cellvibrio japonicus Glycoside Hydrolase Family 31 a-Xylosidase2012In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 18, no 42, p. 13395-13404Article in journal (Refereed)
    Abstract [en]

    The study of the interaction of glycoside hydrolases with their substrates is fundamental to diverse applications in medicine, food and feed production, and biomass-resource utilization. Recent molecular modeling of the a-xylosidase CjXyl31A from the soil saprophyte Cellvibrio japonicus, together with protein crystallography and enzyme-kinetic analysis, has suggested that an appended PA14 protein domain, unique among glycoside hydrolase family 31 members, may confer specificity for large oligosaccharide fragments of the ubiquitous plant polysaccharide xyloglucan (J. Larsbrink, A. Izumi, F. M. Ibatullin, A. Nakhai, H. J. Gilbert, G. J. Davies, H. Brumer, Biochem. J. 2011, 436, 567580). In the present study, a combination of NMR spectroscopic techniques, including saturation transfer difference (STD) and transfer NOE (TR-NOE) spectroscopy, was used to reveal extensive interactions between CjXyl31A active-site variants and xyloglucan hexa- and heptasaccharides. The data specifically indicate that the enzyme recognizes the entire cello-tetraosyl backbone of the substrate and product in positive enzyme subsites and makes further significant interactions with internal pendant a-(1?6)-linked xylosyl units. As such, the present analysis provides an important rationalization of previous kinetic data on CjXyl31A and unique insight into the role of the PA14 domain, which was not otherwise obtainable by protein crystallography.

  • 278. Song, Chunxu
    et al.
    Sundqvist, Gustav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Malm, Erik
    KTH, School of Biotechnology (BIO), Glycoscience.
    de Bruijn, Irene
    Kumar, Aundy
    van de Mortel, Judith
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Raaijmakers, Jos M.
    Lipopeptide biosynthesis in Pseudomonas fluorescens is regulated by the protease complex ClpAP2015In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 15, article id 29Article in journal (Refereed)
    Abstract [en]

    Background: Lipopeptides (LP) are structurally diverse compounds with potent surfactant and broad-spectrum antibiotic activities. In Pseudomonas and other bacterial genera, LP biosynthesis is governed by large multimodular nonribosomal peptide synthetases (NRPS). To date, relatively little is known about the regulatory genetic network of LP biosynthesis. Results: This study provides evidence that the chaperone ClpA, together with the serine protease ClpP, regulates the biosynthesis of the LP massetolide in Pseudomonas fluorescens SS101. Whole-genome transcriptome analyses of clpA and clpP mutants showed their involvement in the transcription of the NRPS genes massABC and the transcriptional regulator massAR. In addition, transcription of genes associated with cell wall and membrane biogenesis, energy production and conversion, amino acid transport and metabolism, and pilus assembly were altered by mutations in clpA and clpP. Proteome analysis allowed the identification of additional cellular changes associated to clpA and clpP mutations. The expression of proteins of the citrate cycle and the heat shock proteins DnaK and DnaJ were particularly affected. Combined with previous findings, these results suggest that the ClpAP complex regulates massetolide biosynthesis via the pathway-specific, LuxR-type regulator MassAR, the heat shock proteins DnaK and DnaJ, and proteins of the TCA cycle. Conclusions: Combining transcriptome and proteome analyses provided new insights into the regulation of LP biosynthesis in P. fluorescens and led to the identification of specific missing links in the regulatory pathways.

  • 279.
    Song, Yajing
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gyarmati, Péter
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Araújo, Ana Catarina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brumer, Harry, III
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Ståhl, Patrik L.
    Visual detection of DNA on paper chips2014In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 86, no 3, p. 1575-1582Article in journal (Refereed)
    Abstract [en]

    On-site DNA analysis for diagnostic or forensic purposes is much anticipated in the future of molecular testing. Yet the challenges to achieve this goal remain large with rapid and inexpensive detection and visualization being key factors for any portable analysis system. We have developed a filter paper-based nucleic acid assay, which is able to identify and distinguish dog and human genomic and mitochondrial samples in a forensic setting. The filter paper material allows for transport by capillary force of the sample DNA through the detection surface, allowing the targets to hybridize specifically to their complementary capture sequences. Coupling micrometer-sized beads to DNA allows the results to be visualized by the naked eye, enabling instant, cost-efficient, and on-site detection, while eliminating the need for advanced expensive instrumentation.

  • 280. Soultani-Vigneron, S.
    et al.
    Dugas, V.
    Rouillat, M. H.
    Fedolliere, J.
    Duclos, M. C.
    Vnuk, E.
    Phaner-Goutorbe, M.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Martin, J. R.
    Wallach, J.
    Cloarec, J. P.
    Immobilisation of oligo-peptidic probes for microarray implementation: Characterisation by FTIR, Atomic Force Microscopy and 2D fluorescence2005In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 822, no 02-jan, p. 304-310Article in journal (Refereed)
    Abstract [en]

    Proteomic microarrays show a wide range of applications for the investigation of DNA-protein, enzyme-substrate as well as protein-protein interactions. Among many challenges to build a viable protein microarray, the surface chemistry that will allow to immobilised various proteins to retain their biological activity is of paramount importance. Here we report a chemical functionalisation method allowing immobilisation of oligo-peptides onto silica surface (porous silica, glass, thermal silicon dioxide). Substrates were first derivatised with a monofunctional silane allowing the elaboration of dense and uniform monolayers in highly reproducible way. Prior to the oligo-peptides grafting, this organic layer was functionalised with an amino-polyethyleneglycol. The coupling step of oligo-peptides onto functionalised supports is achieved through activation of the C-terminal function of the oligo-peptides. Chemical surface modifications were followed by FTIR spectroscopy, AFM measurements and fluorescence scanning microscopy. A systematic study of the oligo-peptide grafting conditions (time, concentration, solvent) was carried out to optimise this step. The oligo-peptides grafting strategy implemented in this work ensure a covalent and oriented grafting of the oligo-peptides. This orientation is ensured through the use of fully protected peptide except the terminal primary an-tine. The immobilized peptides will be then deprotected before biological recognition. This strategy is crucial to retain the biological activity of thousands of oligo-probes assessed on a microarray.

  • 281.
    Spadiut, Oliver
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brugger, Dagmar
    Coman, Vasile
    Haltrich, Dietmar
    Gorton, Lo
    Engineered Pyranose 2-Oxidase: Efficiently Turning Sugars into Electrical Energy2010In: Electroanalysis, ISSN 1040-0397, E-ISSN 1521-4109, Vol. 22, no 7-8, p. 813-820Article in journal (Refereed)
    Abstract [en]

    Due to the recent interest in enzymatic biofuel cells (BECs), sugar oxidizing enzymes other than the commonly used glucose oxidase are gaining more importance as possible bioelements of implantable microscale-devices, which can, for example, be used in biosensors and pacemakers. In this study we used rational and semi-rational protein design to improve the catalytic activity of the enzyme pyranose 2-oxidase (P2Ox) with its alternative soluble electron acceptors 1,4-benzoquinone and ferricenium ion, which can serve as electron mediators, to possibly boost the power output of enzymatic BECs. Using a screening assay based on 96-well plates, we identified the variant H450G, which showed lower K-M and higher k(cat) values for both 1,4-benzoquinone and ferricenium ion compared to the wild-type enzyme, when either D-glucose or D-galactose were used as saturating electron donors. Besides this variant, we analyzed the variants V546C and T169G/V546C for their possible application in enzymatic BECs. The results obtained in homogeneous solution were compared with those obtained when P2Ox was immobilized on the surface of graphite electrodes and either "wired" to an osmium redox polymer or using soluble 1,4-benzoquinone as mediator. According to the spectrophotometrically determined kinetic constants, the possible energy output, measured in flow injection analysis experiments with these variants, increased up to 4-fold compared to systems employing the wild-type enzyme. Our results show that by increasing the catalytic activity of the redox enzyme P2Ox with its alternative electron acceptors 1,4-benzoquinone and ferricenium ion, it is possible to achieve a higher energy output of an enzymatic BFC when using the same concentration of sugar substrate.

  • 282.
    Spadiut, Oliver
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Ibatullin, Farid M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Peart, Jonelle
    KTH, School of Biotechnology (BIO), Glycoscience.
    Gullfot, Fredrika
    KTH, School of Biotechnology (BIO), Glycoscience.
    Martinez-Fleites, Carlos
    Ruda, Marcus
    Xu, Chunlin
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Sundqvist, Gustav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Davies, Gideon J.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Building Custom Polysaccharides in Vitro with an Efficient, Broad-Specificity Xyloglucan Glycosynthase and a Fucosyltransferase2011In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 133, no 28, p. 10892-10900Article in journal (Refereed)
    Abstract [en]

    The current drive for applications of biomass-derived compounds, for energy and advanced materials, has led to a resurgence of interest in the manipulation of plant polymers. The xyloglucans, a family of structurally complex plant polysaccharides, have attracted significant interest due to their intrinsic high affinity for cellulose, both in muro and in technical applications. Moreover, current cell wall models are limited by the lack of detailed structure-property relationships of xyloglucans, due to a lack of molecules with well-defined branching patterns. Here, we have developed a new, broad-specificity "xyloglucan glycosynthase", selected from active-site mutants of a bacterial endoxyloglucanase, which catalyzed the synthesis of high molar mass polysaccharides, with complex side-chain structures, from suitable glycosyl fluoride donor substrates. The product range was further extended by combination with an Arabidopsis thaliana alpha(1 -> 2)-fucosyltransferase to achieve the in vitro synthesis of fucosylated xyloglucans typical of dicot primary cell walls. These enzymes thus comprise a toolkit for the controlled enzymatic synthesis of xyloglucans that are otherwise impossible to obtain from native sources. Moreover, this study demonstrates the validity of a chemo-enzymatic approach to polysaccharide synthesis, in which the simplicity and economy of glycosynthase technology is harnessed together with the exquisite specificity of glycosyltransferases to control molecular complexity.

  • 283. Spadiut, Oliver
    et al.
    Leitner, Christian
    Salaheddin, Clara
    Varga, Balazs
    Vertessy, Beata G.
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Glycoscience.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Haltrich, Dietmar
    Improving thermostability and catalytic activity of pyranose 2-oxidase from Trametes multicolor by rational and semi-rational design2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 3, p. 776-792Article in journal (Refereed)
    Abstract [en]

    The fungal homotetrameric flavoprotein pyranose 2-oxidase (P2Ox; EC 1.1.3.10) catalyses the oxidation of various sugars at position C2, while, concomitantly, electrons are transferred to oxygen as well as to alternative electron acceptors (e.g. oxidized ferrocenes). These properties make P2Ox an interesting enzyme for various biotechnological applications. Random mutagenesis has previously been used to identify variant E542K, which shows increased thermostability. In the present study, we selected position Leu537 for saturation mutagenesis, and identified variants L537G and L537W, which are characterized by a higher stability and improved catalytic properties. We report detailed studies on both thermodynamic and kinetic stability, as well as the kinetic properties of the mutational variants E542K, E542R, L537G and L537W, and the respective double mutants (L537G/E542K, L537G/E542R, L537W/E542K and L537W/E542R). The selected substitutions at positions Leu537 and Glu542 increase the melting temperature by approximately 10 and 14 degrees C, respectively, relative to the wild-type enzyme. Although both wild-type and single mutants showed first-order inactivation kinetics, thermal unfolding and inactivation was more complex for the double mutants, showing two distinct phases, as revealed by microcalorimetry and CD spectroscopy. Structural information on the variants does not provide a definitive answer with respect to the stabilizing effects or the alteration of the unfolding process. Distinct differences, however, are observed for the P2Ox Leu537 variants at the interfaces between the subunits, which results in tighter association.

  • 284. Spadiut, Oliver
    et al.
    Leitner, Christian
    Tan, Tien-Chye
    Ludwig, Roland
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Haltrich, Dietmar
    Mutations of Thr169 affect substrate specificity of pyranose 2-oxidase from Trametes multicolor2008In: Biocatalysis and Biotransformation, ISSN 1024-2422, E-ISSN 1029-2446, Vol. 26, no 1-2, p. 120-127Article in journal (Refereed)
    Abstract [en]

    Site-directed mutagenesis was used to enhance the catalytic activity of pyranose 2-oxidase (P2Ox) from Trametes multicolor with different substrates. To this end, threonine at position 169 was replaced by glycine, alanine and serine, respectively. Using oxygen as electron acceptor the mutant T169G was equally active with d-glucose and d-galactose, whereas wild-type recombinant P2Ox only showed 5.2% relative activity with the latter substrate. When d-galactose was used as electron donor in saturating concentrations, T169G showed a 4.5-fold increase in its catalytic efficiency k(cat)/K-M for the alternative electron acceptor 1,4-benzoquinone and a nine-fold increased k(cat)/K-M value with the ferricenium ion compared with wt recP2Ox. Variant T169S showed an increase in its catalytic efficiency both with 1,4-benzoquinone (3.7 times) as well as with the ferricenium ion (1.4 times) when D-glucose was the substrate.

  • 285.
    Spadiut, Oliver
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Olsson, Lisbeth
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    A comparative summary of expression systems for the recombinant production of galactose oxidase2010In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 9, p. 68-Article in journal (Refereed)
    Abstract [en]

    Background: The microbes Escherichia coli and Pichia pastoris are convenient prokaryotic and eukaryotic hosts, respectively, for the recombinant production of proteins at laboratory scales. A comparative study was performed to evaluate a range of constructs and process parameters for the heterologous intra-and extracellular expression of genes encoding the industrially relevant enzyme galactose 6-oxidase (EC 1.1.3.9) from the fungus Fusarium graminearum. In particular, the wild-type galox gene from F. graminearum, an optimized variant for E. coli and a codon-optimized gene for P. pastoris were expressed without the native pro-sequence Results: The intracellular expression of a codon-optimized gene with an N-terminal His(10)-tag in E. coli, using the pET16b(+) vector and BL21DE3 cells, resulted in a volumetric productivity of 180 U.L-1.h(-1). The intracellular expression of the wild-type gene from F. graminearum, using the pPIC3.5 vector and the P. pastoris strain GS115, was poor, resulting in a volumetric productivity of 120 U.L-1.h(-1). Furthermore, this system did not tolerate an N-terminal His(10)-tag, thus rendering isolation of the enzyme from the complicated mixture difficult. The highest volumetric productivity (610 U.L-1.h(-1)) was achieved when the wild-type gene from F. graminearum was expressed extracellularly in the P. pastoris strain SMD1168H using the pPICZ alpha-system. A C-terminal His(6)-tag did not significantly affect the production of the enzyme, thus enabling simple purification by immobilized metal ion affinity chromatography. Notably, codon-optimisation of the galox gene for expression in P. pastoris did not result in a higher product yield (g protein.L-1 culture). Effective activation of the enzyme to generate the active-site radical copper complex could be equally well achieved by addition of CuSO4 directly in the culture medium or post-harvest. Conclusions: The results indicate that intracellular production in E. coli and extracellular production in P. pastoris comprise a complementary pair of systems for the production of GalOx. The prokaryotic host is favored for high-throughput screening, for example in the development of improved enzymes, while the yeast system is ideal for production scale-up for enzyme applications.

  • 286. Spadiut, Oliver
    et al.
    Radakovits, Katrin
    Pisanelli, Ines
    Salaheddin, Clara
    Yamabhai, Montarop
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Glycoscience.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Haltrich, Dietmar
    A thermostable triple mutant of pyranose 2-oxidase from Trametes multicolor with improved properties for biotechnological applications.2009In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 4, no 4, p. 525-534Article in journal (Refereed)
    Abstract [en]

    In order to increase the thermal stability and the catalytic properties of pyranose oxidase (P2Ox) from Trametes multicolor toward its poor substrate D-galactose and the alternative electron acceptor 1,4-benzoquinone (1,4-BQ), we designed the triple-mutant T169G/E542K/V546C. Whereas the wild-type enzyme clearly favors D-glucose as its substrate over D-galactose [substrate selectivity (k(cat)/K(M))(Glc)/(k(cat)/K(M))(Gal) = 172], the variant oxidizes both sugars equally well [(k(cat)/K(M))(Glc)/(k(cat)/K(M))(Gal) = 0.69], which is of interest for food biotechnology. Furthermore, the variant showed lower K(M) values and approximately ten-fold higher k(cat) values for 1,4-BQ when D-galactose was used as the saturating sugar substrate, which makes this enzyme particularly attractive for use in biofuel cells and enzyme-based biosensors. In addition to the altered substrate specificity and reactivity, this mutant also shows significantly improved thermal stability. The half life time at 60 degrees C was approximately 10 h, compared to 7.6 min for the wild-type enzyme. We performed successfully small-scale bioreactor pilot conversion experiments of D-glucose/D-galactose mixtures at both 30 and 50 degrees C, showing the usefulness of this P2Ox variant in biocatalysis as well as the enhanced thermal stability of the enzyme. Moreover, we determined the crystal structure of the mutant in its unligated form at 1.55 A resolution. Modeling D-galactose in position for oxidation at C2 into the mutant active site shows that substituting Thr for Gly at position 169 favorably accommodates the axial C4 hydroxyl group that would otherwise clash with Thr169 in the wild-type.

  • 287.
    Spadiut, Oliver
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Biochemistry.
    Pisanelli, Ines
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO), Biochemistry.
    Importance of the gating segment in the substrate-recognition loop of pyranose 2-oxidase2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 13, p. 2892-2909Article in journal (Refereed)
    Abstract [en]

    Pyranose 2-oxidase from Trametes multicolor is a 270 kDa homotetrameric enzyme that participates in lignocellulose degradation by wood-rotting fungi and oxidizes a variety of aldopyranoses present in lignocellulose to 2-ketoaldoses. The active site in pyranose 2-oxidase is gated by a highly conserved, conformationally degenerate loop (residues 450-461), with a conformer ensemble that can accommodate efficient binding of both electron-donor substrate (sugar) and electron-acceptor substrate (oxygen or quinone compounds) relevant to the sequential reductive and oxidative half-reactions, respectively. To investigate the importance of individual residues in this loop, a systematic mutagenesis approach was used, including alanine-scanning, site-saturation and deletion mutagenesis, and selected variants were characterized by biochemical and crystal-structure analyses. We show that the gating segment (454FSY456) of this loop is particularly important for substrate specificity, discrimination of sugar substrates, turnover half-life and resistance to thermal unfolding, and that three conserved residues (Asp452, Phe454 and Tyr456) are essentially intolerant to substitution. We furthermore propose that the gating segment is of specific importance for the oxidative half-reaction of pyranose 2-oxidase when oxygen is the electron acceptor. Although the position and orientation of the slow substrate 2-deoxy-2-fluoro-glucose when bound in the active site of pyranose 2-oxidase variants is identical to that observed earlier, the substrate-recognition loop in F454N and Y456W displays a high degree of conformational disorder. The present study also lends support to the hypothesis that 1,4-benzoquinone is a physiologically relevant alternative electron acceptor in the oxidative half-reaction.

  • 288.
    Srivastava, Vaibhav
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Malm, Erik
    KTH, School of Biotechnology (BIO), Glycoscience.
    Sundqvist, Gustav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Quantitative Proteomics Reveals that Plasma Membrane Microdomains From Poplar Cell Suspension Cultures Are Enriched in Markers of Signal Transduction, Molecular Transport, and Callose Biosynthesis2013In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, no 12, p. 3874-3885Article in journal (Refereed)
    Abstract [en]

    The plasma membrane (PM) is a highly dynamic interface that contains detergent-resistant microdomains (DRMs). The aim of this work was to determine the main functions of such microdomains in poplar through a proteomic analysis using gel-based and solution (iTRAQ) approaches. A total of 80 proteins from a limited number of functional classes were found to be significantly enriched in DRM relative to PM. The enriched proteins are markers of signal transduction, molecular transport at the PM, or cell wall biosynthesis. Their intrinsic properties are presented and discussed together with the biological significance of their enrichment in DRM. Of particular importance is the significant and specific enrichment of several callose [(1→3)-β-glucan] synthase isoforms, whose catalytic activity represents a final response to stress, leading to the deposition of callose plugs at the surface of the PM. An integrated functional model that connects all DRM-enriched proteins identified is proposed. This report is the only quantitative analysis available to date of the protein composition of membrane microdomains from a tree species.

  • 289.
    Srivastava, Vaibhav
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Obudulu, O.
    Bygdell, J.
    Löfstedt, T.
    Rydén, P.
    Nilsson, R.
    Ahnlund, M.
    Johansson, A.
    Jonsson, P.
    Freyhult, E.
    Qvarnström, J.
    Karlsson, J.
    Melzer, M.
    Moritz, T.
    Trygg, J.
    Hvidsten, T. R.
    Wingsle, G.
    OnPLS integration of transcriptomic, proteomic and metabolomic data shows multi-level oxidative stress responses in the cambium of transgenic hipI- superoxide dismutase Populus plants2013In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 14, no 1, p. 893-Article in journal (Refereed)
    Abstract [en]

    Background: Reactive oxygen species (ROS) are involved in the regulation of diverse physiological processes in plants, including various biotic and abiotic stress responses. Thus, oxidative stress tolerance mechanisms in plants are complex, and diverse responses at multiple levels need to be characterized in order to understand them. Here we present system responses to oxidative stress in Populus by integrating data from analyses of the cambial region of wild-type controls and plants expressing high-isoelectric-point superoxide dismutase (hipI-SOD) transcripts in antisense orientation showing a higher production of superoxide. The cambium, a thin cell layer, generates cells that differentiate to form either phloem or xylem and is hypothesized to be a major reason for phenotypic perturbations in the transgenic plants. Data from multiple platforms including transcriptomics (microarray analysis), proteomics (UPLC/QTOF-MS), and metabolomics (GC-TOF/MS, UPLC/MS, and UHPLC-LTQ/MS) were integrated using the most recent development of orthogonal projections to latent structures called OnPLS. OnPLS is a symmetrical multi-block method that does not depend on the order of analysis when more than two blocks are analysed. Significantly affected genes, proteins and metabolites were then visualized in painted pathway diagrams. Results: The main categories that appear to be significantly influenced in the transgenic plants were pathways related to redox regulation, carbon metabolism and protein degradation, e.g. the glycolysis and pentose phosphate pathways (PPP). The results provide system-level information on ROS metabolism and responses to oxidative stress, and indicate that some initial responses to oxidative stress may share common pathways.Conclusion: The proposed data evaluation strategy shows an efficient way of compiling complex, multi-platform datasets to obtain significant biological information.

  • 290.
    Srivastava, Vaibhav
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Weber, Joseph R.
    Malm, Erik
    Fouke, Bruce W.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Proteomic Analysis of a Poplar Cell Suspension Culture Suggests a Major Role of Protein S-Acylation in Diverse Cellular Processes2016In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 7, article id 477Article in journal (Refereed)
    Abstract [en]

    S-acylation is a reversible post-translational modification of proteins known to be involved in membrane targeting, subcellular trafficking, and the determination of a great variety of functional properties of proteins. The aim of this work was to identify S-acylated proteins in poplar. The use of an acyl-biotin exchange method and mass spectrometry allowed the identification of around 450 S-acylated proteins, which were subdivided into three major groups of proteins involved in transport, signal transduction, and response to stress, respectively. The largest group of S-acylated proteins was the protein kinase superfamily. Soluble N-ethylmaleimide-sensitive factor-activating protein receptors, band 7 family proteins and tetraspanins, all primarily related to intracellular trafficking, were also identified. In addition, cell wall related proteins, including cellulose synthases and other glucan synthases, were found to be S-acylated. Twenty four of the identified S-acylated proteins were also enriched in detergent-resistant membrane microdomains, suggesting S-acylation plays a key role in the localization of proteins to specialized plasma membrane subdomains. This dataset promises to enhance our current understanding of the various functions of S-acylated proteins in plants.

  • 291. Steiner, Eva Maria
    et al.
    Both, Dominic
    Lossl, Philip
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience.
    Schnell, Robert
    Schneider, Gunter
    CysK2 from Mycobacterium tuberculosis Is an O-Phospho-L-Serine-Dependent S-Sulfocysteine Synthase2014In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 196, no 19, p. 3410-3420Article in journal (Refereed)
    Abstract [en]

    Mycobacterium tuberculosis is dependent on cysteine biosynthesis, and reduced sulfur compounds such as mycothiol synthesized from cysteine serve in first-line defense mechanisms against oxidative stress imposed by macrophages. Two biosynthetic routes to L-cysteine, each with its own specific cysteine synthase (CysK1 and CysM), have been described in M. tuberculosis, but the function of a third putative sulfhydrylase in this pathogen, CysK2, has remained elusive. We present biochemical and biophysical evidence that CysK2 is an S-sulfocysteine synthase, utilizing O-phosphoserine (OPS) and thiosulfate as substrates. The enzyme uses a mechanism via a central aminoacrylate intermediate that is similar to that of other members of this pyridoxal phosphate-dependent enzyme family. The apparent second-order rate of the first half-reaction with OPS was determined as k(max)/K-s = (3.97 x 10(3)) +/- 619 M-1 s(-1), which compares well to the OPS-specific mycobacterial cysteine synthase CysM with a k(max)/K-s of (1.34 x 10(3)) +/- 48.2. Notably, CysK2 does not utilize thiocarboxylated CysO as a sulfur donor but accepts thiosulfate and sulfide as donor substrates. The specificity constant k(cat)/K-m for thiosulfate is 40-fold higher than for sulfide, suggesting an annotation as S-sulfocysteine synthase. Mycobacterial CysK2 thus provides a third metabolic route to cysteine, either directly using sulfide as donor or indirectly via S-sulfocysteine. Hypothetically, S-sulfocysteine could also act as a signaling molecule triggering additional responses in redox defense in the pathogen upon exposure to reactive oxygen species during dormancy.

  • 292. Stepper, Judith
    et al.
    Dabin, Jerome
    Eklöf, Jens M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Thongpoo, Preeyanuch
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kongsaeree, Prachumporn
    Taylor, Edward J.
    Turkenburg, Johan P.
    Brumer, Harry
    University of British Columbia, Canada.
    Davies, Gideon J.
    Structure and activity of the Streptococcus pyogenes family GH1 6-phospho-beta-glucosidase SPy15992013In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 69, p. 16-23Article in journal (Refereed)
    Abstract [en]

    The group A streptococcus Streptococcus pyogenes is the causative agent of a wide spectrum of invasive infections, including necrotizing fasciitis, scarlet fever and toxic shock syndrome. In the context of its carbohydrate chemistry, it is interesting that S. pyogenes (in this work strain M1 GAS SF370) displays a spectrum of oligosaccharide-processing enzymes that are located in close proximity on the genome but that the in vivo function of these proteins remains unknown. These proteins include different sugar transporters (SPy1593 and SPy1595), both GH125 alpha-1,6- and GH38 alpha-1,3-mannosidases (SPy1603 and SPy1604), a GH84 beta-hexosaminidase (SPy1600) and a putative GH2 beta-galactosidase (SPy1586), as well as SPy1599, a family GH1 'putative beta-glucosidase'. Here, the solution of the three-dimensional structure of SPy1599 in a number of crystal forms complicated by unusual crystallographic twinning is reported. The structure is a classical (beta/alpha)(g)-barrel, consistent with CAZy family GH1 and other members of the GH-A clan. SPy1599 has been annotated in sequence depositions as a beta-glucosidase (EC 3.2.1.21), but no such activity could be found; instead, three-dimensional structural overlaps with other enzymes of known function suggested that SPy1599 contains a phosphate-binding pocket in the active site and has possible 6-phospho-beta-glycosidase activity. Subsequent kinetic analysis indeed showed that SPy1599 has 6-phospho-beta-glucosidase (EC 3.2.1.86) activity. These data suggest that SPy1599 is involved in the intracellular degradation of 6-phosphoglycosides, which are likely to originate from import through one of the organism's many phosphoenolpyruvate phosphotransfer systems (PEP-PTSs).

  • 293. Ståhl, Patrik
    et al.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Glycoscience.
    Araújo Silva, Ana Catarina
    Song, Yajing
    Surface-functionalized cellulosic fibres, method of manufacture thereof and uses thereof2011Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    A surface-functionalized cellulosic fibre, characterized in that a functional group denoted X is covalently linked to cellulose with a linkage having structure: X-(NH)-(C=S)-0-cellulose molecule, optionally further comprising xyloglucan. Methods of manufacture thereof and uses thereof in analytical methods.

  • 294. Sullivan, Mitchell A.
    et al.
    Powell, Prudence O.
    Witt, Torsten
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Roura, Eugeni
    Gilbert, Robert G.
    Improving size-exclusion chromatography separation for glycogen2014In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1332, p. 21-29Article in journal (Refereed)
    Abstract [en]

    Glycogen is a hyperbranched glucose polymer comprised of glycogen beta particles, which can also form much larger composite alpha particles. The recent discovery using size-exclusion chromatography (SEC) that fewer, smaller, alpha particles are found in diabetic-mouse liver compared to healthy mice highlights the need to achieve greater accuracy in the size separation methods used to analyze alpha and beta particles. While past studies have used dimethyl sulfoxide as the SEC eluent to analyze the molecular size and structure of native glycogen, an aqueous eluent has not been rigorously tested and compared with dimethyl sulfoxide. The conditions for SEC of pig-liver glycogen, phytoglycogen and oyster glycogen were optimized by comparing two different eluents, aqueous 50 mM NH4NO3/0.02% NaN3 and dimethyl sulfoxide/0.5% LiBr, run through different column materials and pore sizes at various flow rates. The aqueous system gave distinct size separation of alpha- and beta-particle peaks, allowing for a more detailed and quantitative analysis and comparison between liver glycogen samples. This greater resolution has also revealed key differences between the structure of liver glycogen and phytoglycogen.

  • 295.
    Sundqvist, Gustav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Analysis of noncovalent and covalent protein-ligand complexes by electrospray ionisation mass spectrometry2008Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    In this thesis, the application of electrospray ionisation mass spectrometry (ESI-MS) to the analysis of intact proteins is demonstrated. In papers I and II, the use of ESI-MS for the analysis of noncovalent protein-ligand complexes were discussed. In addition, the interfacing of liquid chromatography (LC) with ESI-MS and the development of an LC-ESI-MS method were demonstrated in paper III for the quality control of recombinant proteins. Furthermore, this method was applied in paper IV for the analysis of covalent glycosyl-enzyme intermediates.

    The monitoring of noncovalent complexes by ESI-MS is well established. However, the varying characteristic of ESI-MS data, especially in the analysis of noncovalent complexes can make the quantification of such complexes troublesome. In paper I, it was demonstrated how the variation in the position of the ESI-emitter and the initial droplet size of the electrosprayed droplets, together with different partitioning of a protein and its ligand in these droplets, can be the cause of such varying characteristics. Furthermore, it was shown that the partitioning can be of electrostatic and/or hydrophobic/hydrophilic origin. Thus it was demonstrated that if the ligand is more hydrophobic and thereby more surface active relative to the protein, decreasing the droplet size or increasing the distance between the electrospray emitter and the sampling orifice will lead to more efficient sampling of the droplet bulk where the ligand concentration is low. This results in a favoured sampling of free protein relative to the protein ligand complex. The opposite was shown to occur if the ligand is more hydrophilic than the protein.

    In paper II, Ribonuclease A (RNAse) was used as a model for enzymes acting on polymeric substrates with different chain lengths. Nano-ESI-MS was applied to monitor the noncovalent interactions between RNAse and different target ligands. Among the single building blocks of RNA, including ribose, the bases adenine, guanine, cytosine and uracil, and phosphate, only phosphate was observed to interact at multiple RNAse sites at a higher cone voltage. Furthermore, monobasic singlestranded deoxycytidylic acid oligomers (dCx) of different lengths (X=6, 9 and 12), and RNAse were analysed with nano-ESI-MS. The deoxycytidylic acid with 12 nucleotides was observed with the highest complex to free protein ratio, hence indicating the strongest interaction. Finally, collision induced dissociation of the noncovalent RNAseA-dC6 complex resulted in dissociation of covalently bound cytosine from the nucleotide backbone rather than break up of the noncovalent complex, illustrating the cooperative effect of multiple noncovalent interactions.

    In paper III an LC-ESI-MS method was presented capable of analysing proteins 10-100 kDa in size, from salt-containing liquid samples. The proteins included human protein fragments for the largescale production of antibodies and human protein targets for structural determination, expressed in E. coli. Also, glycosylated proteins expressed in Pichia pastoris were analysed. The method provides fast chromatography, is robust and makes use of cheap desalting/trap columns. In addition it was used with optimised reduction and alkylation protocols in order to minimize protein aggregation of denatured and incorrectly folded proteins containing cysteins, which otherwise form adducts by disulfide bond formation. Furthermore, the method was used in paper IV for the quantification of covalent proteinligand intermediates formed enzymatically between PttXET16-34, a xyloglucan endo-transglycosylase (XET) from hybrid aspen, and the synthetic substrates GalGXXXGGG and GalXXXGXXXG designed in order to function as donor substrates only. Thus covalent GalG-enzyme and GalGXXXG-enzyme complexes were detected. Moreover, establishing of a pseudo equilibrium for the formation of the covalent GalGXXXG-enzyme complex enabled quantification of the saccharide and enzyme constituents of this equilibrium and determination of the free energy of formation (∆G0). The high mass resolution of the TOF-MS allowed unambiguous assessment of the covalent nature of the glycosyl-enzyme complexes. Morover, the formation of noncovalent complexes between excess substrate and protein, which can deteriorate MS-signal and increase spectrum complexity, was efficiently avoided by the chromatographic step, which separated the saccharide content from the protein content.

  • 296.
    Sundqvist, Gustav
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Stenvall, Maria
    KTH, School of Biotechnology (BIO).
    Berglund, Helena
    Ottosson, Jenny
    KTH, School of Biotechnology (BIO).
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    A general, robust method for the quality control of intact proteins using LC–ESI-MS2007In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 852, no 1-2, p. 188-194Article in journal (Refereed)
    Abstract [en]

    A simple and robust method for the routine quality control of intact proteins based on liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) is presented. A wide range of prokaryotic and eukaryotic proteins expressed recombinantly in Escherichia coli or Pichia pastoris has been analyzed with medium- to high-throughput with on-line desalting from multi-well sample plates. Particular advantages of the method include fast chromatography and short cycle times, the use of inexpensive trapping/desalting columns, low sample carryover, and the ability to analyze proteins with masses ranging from 5 to 100 kDa with greater than 50 ppm accuracy. Moreover, the method can be readily coupled with optimized chemical reduction and alkylation steps to facilitate the analysis of denatured or incorrectly folded proteins (e.g., recombinant proteins sequestered in E. coli inclusion bodies) bearing cysteine residues, which otherwise form intractable multimers and non-specific adducts by disulfide bond formation.

  • 297. Svagan, A. J.
    et al.
    Kusic, A.
    De Gobba, C.
    Larsen, F. H.
    Sassene, P.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Van De Weert, M.
    Mullertz, A.
    Jørgensen, B.
    Ulvskov, P.
    Rhamnogalacturonan-I based microcapsules for targeted drug release2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 12, article id e0168050Article in journal (Refereed)
    Abstract [en]

    Drug targeting to the colon via the oral administration route for local treatment of e.g. inflammatory bowel disease and colonic cancer has several advantages such as needle-free administration and low infection risk. A new source for delivery is plant-polysaccharide based delivery platforms such as Rhamnogalacturonan-I (RG-I). In the gastro-intestinal tract the RG-I is only degraded by the action of the colonic microflora. For assessment of potential drug delivery properties, RG-I based microcapsules (~1 μm in diameter) were prepared by an interfacial poly-addition reaction. The cross-linked capsules were loaded with a fluorescent dye (model drug). The capsules showed negligible and very little in vitro release when subjected to media simulating gastric and intestinal fluids, respectively. However, upon exposure to a cocktail of commercial RG-I cleaving enzymes, ~ 9 times higher release was observed, demonstrating that the capsules can be opened by enzymatic degradation. The combined results suggest a potential platform for targeted drug delivery in the terminal gastro-intestinal tract.

  • 298. Takahashi, Junko
    et al.
    Awano, Tatsuya
    Winzell, Anders
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kallas, Åsa
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ratke, Christine
    Gorzsas, Andras
    Lesniewska, Joanna
    Anne, Gouget
    Berthold, Fredrik
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Sundberg, Björn
    Mellerowicz, Eva
    Suppression of wood expressed xylanase affects cell expansion and secondary wall compositionManuscript (preprint) (Other academic)
  • 299.
    Tan, Tien Chye
    et al.
    Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience.
    Gandini, Rosaria
    Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Structural Basis for Binding of Fluorinated Glucose and Galactose to Trametes multicolor Pyranose 2-Oxidase Variants with Improved Galactose Conversion2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 1, article id e86736Article in journal (Refereed)
    Abstract [en]

    Each year, about six million tons of lactose are generated from liquid whey as industrial byproduct, and optimally this large carbohydrate waste should be used for the production of value-added products. Trametes multicolor pyranose 2-oxidase (TmP2O) catalyzes the oxidation of various monosaccharides to the corresponding 2-keto sugars. Thus, a potential use of TmP2O is to convert the products from lactose hydrolysis, D-glucose and D-galactose, to more valuable products such as tagatose. Oxidation of glucose is however strongly favored over galactose, and oxidation of both substrates at more equal rates is desirable. Characterization of TmP2O variants (H450G, V546C, H450G/ V546C) with improved D-galactose conversion has been given earlier, of which H450G displayed the best relative conversion between the substrates. To rationalize the changes in conversion rates, we have analyzed high-resolution crystal structures of the aforementioned mutants with bound 2- and 3-fluorinated glucose and galactose. Binding of glucose and galactose in the productive 2-oxidation binding mode is nearly identical in all mutants, suggesting that this binding mode is essentially unaffected by the mutations. For the competing glucose binding mode, enzyme variants carrying the H450G replacement stabilize glucose as the a-anomer in position for 3-oxidation. The backbone relaxation at position 450 allows the substrate-binding loop to fold tightly around the ligand. V546C however stabilize glucose as the beta-anomer using an open loop conformation. Improved binding of galactose is enabled by subtle relaxation effects at key active-site backbone positions. The competing binding mode for galactose 2-oxidation by V546C stabilizes the beta-anomer for oxidation at C1, whereas H450G variants stabilize the 3-oxidation binding mode of the galactose alpha-anomer. The present study provides a detailed description of binding modes that rationalize changes in the relative conversion rates of D-glucose and D-galactose and can be used to refine future enzyme designs for more efficient use of lactose-hydrolysis byproducts.

  • 300.
    Tan, Tien Chye
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience.
    Wongnate, T.
    Sucharitakul, J.
    Krondorfer, I.
    Sygmund, C.
    Haltrich, D.
    Chaiyen, P.
    Peterbauer, C. K.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    The 1.6 Å Crystal Structure of Pyranose Dehydrogenase from Agaricus meleagris Rationalizes Substrate Specificity and Reveals a Flavin Intermediate2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 1Article in journal (Refereed)
    Abstract [en]

    Pyranose dehydrogenases (PDHs) are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a) position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O) activates oxygen by a mechanism that proceeds via a covalent flavin C(4a)-hydroperoxide intermediate. Although the flavin C(4a) adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2 position accessible for oxidation, whereas 2-fluorinated glucose performed poorly (C3 accessible), indicating that the glucose C2 position is the primary site of attack.

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