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  • 1.
    Aspeborg, Henrik
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bhalerao, Rishikeshi
    Hertzberg, Magnus
    Johansson, Karin
    Johnsson, P.
    Karlsson, Ann
    Sandberg, Göran
    Schrader, Jarmo
    Sundberg, Björn
    Teeri, Tuula
    Trygg, Johan
    Wallbäcks, Lars
    Vegetabile material, plants and a method of producing a plant having altered lignin properties2008Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    The present invention is related to a set of genes, which when modified in plants gives altered lignin properties. The invention provides DNA construct such as a vector useful in the method of the invention. Further, the invention relates to a plant cell or plant progeny of the plants and wood produced by the plants according to the invention Lower lignin levels will result in improved saccharification for bio-refining and ethanol production and improved pulp and paper. Increased lignin levels will utilise lignin properties for energy production. The genes and DNA constructs may be used for the identification of plants having altered lignin characteristics as compared to the wild-type. According to the invention genes and DNA constructs may also be used as candidate genes in marker assisted breeding.

  • 2.
    Aspeborg, Henrik
    et al.
    KTH, School of Biotechnology (BIO).
    Schrader, J.
    Coutinho, P. M.
    Stam, M.
    Kallas, A.
    Djerbi, S.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Denman, S.
    Amini, B.
    Sterky, Fredrik
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Master, E.
    Sandberg, G.
    Mellerowicz, E.
    Sundberg, B.
    Henrissat, B.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Carbohydrate-active enzymes involved in the secondary cell wall biogenesis in hybrid aspen2005In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 137, no 3, 983-997 p.Article in journal (Refereed)
    Abstract [en]

    Wood formation is a fundamental biological process with significant economic interest. While lignin biosynthesis is currently relatively well understood, the pathways leading to the synthesis of the key structural carbohydrates in wood fibers remain obscure. We have used a functional genomics approach to identify enzymes involved in carbohydrate biosynthesis and remodeling during xylem development in the hybrid aspen Populus tremula x tremuloides. Microarrays containing cDNA clones from different tissue-specific libraries were hybridized with probes obtained from narrow tissue sections prepared by cryosectioning of the developing xylem. Bioinformatic analyses using the sensitive tools developed for carbohydrate-active enzymes allowed the identification of 25 xylem-specific glycosyltransferases belonging to the Carbohydrate-Active EnZYme families GT2, GT8, GT14, GT31, GT43, GT47, and GT61 and nine glycosidases (or transglycosidases) belonging to the Carbohydrate-Active EnZYme families GH9, GH10, GH16, GH17, GH19, GH28, GH35, and GH51. While no genes encoding either polysaccharide lyases or carbohydrate esterases were found among the secondary wall-specific genes, one putative O-acetyltransferase was identified. These wood-specific enzyme genes constitute a valuable resource for future development of engineered fibers with improved performance in different applications.

  • 3.
    Baumann, Martin J.
    et al.
    KTH, School of Biotechnology (BIO).
    Eklöf, Jens M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Michel, Gurvan
    Kallas, Åsa M.
    KTH, School of Biotechnology (BIO).
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Czjzek, Mirjam
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: Biological implications for cell wall metabolism2007In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 19, no 6, 1947-1963 p.Article in journal (Refereed)
    Abstract [en]

    High-resolution, three-dimensional structures of the archetypal glycoside hydrolase family 16 (GH16) endo-xyloglucanases Tm-NXG1 and Tm-NXG2 from nasturtium (Tropaeolum majus) have been solved by x-ray crystallography. Key structural features that modulate the relative rates of substrate hydrolysis to transglycosylation in the GH16 xyloglucan-active enzymes were identified by structure-function studies of the recombinantly expressed enzymes in comparison with data for the strict xyloglucan endo-transglycosylase Ptt-XET16-34 from hybrid aspen ( Populus tremula 3 Populus tremuloides). Production of the loop deletion variant Tm-NXG1-Delta YNIIG yielded an enzyme that was structurally similar to Ptt- XET16-34 and had a greatly increased transglycosylation: hydrolysis ratio. Comprehensive bioinformatic analyses of XTH gene products, together with detailed kinetic data, strongly suggest that xyloglucanase activity has evolved as a gain of function in an ancestral GH16 XET to meet specific biological requirements during seed germination, fruit ripening, and rapid wall expansion.

  • 4.
    Baumann, Martin J.
    et al.
    KTH, School of Biotechnology (BIO).
    Eklöf, Jens
    KTH, School of Biotechnology (BIO).
    Michel, G.
    Kallas, Åsa
    KTH.
    Teeri, Tuula
    KTH, School of Biotechnology (BIO).
    Czjzek, Mirjam
    KTH.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Structural analysis of nasturtium NXG reveals the evolution of GH16 xyloglucanase activity from XETs: biological implications for cell wall metabolismManuscript (Other academic)
  • 5. Becker, D.
    et al.
    Braet, C.
    Brumer, Harry
    KTH, Superseded Departments, Biotechnology.
    Claeyssens, M.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    Fagerstrom, B. R.
    Harris, M.
    Jones, T. A.
    Kleywegt, G. J.
    Koivula, A.
    Mahdi, S.
    Piens, K.
    Sinnott, M. L.
    Stahlberg, J.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Underwood, M.
    Wohlfahrt, G.
    Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei CeI7A and its E223S/A224H/L225V/T226A/D262G mutant2001In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 356, 19-30 p.Article in journal (Refereed)
    Abstract [en]

    The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala(224)) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves, The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 Angstrom (= 0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 Angstrom contact between N-2 and O'(1). The pH variation of k(cat)/K-m for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wildtype and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) shifting from 5.99+/-0.02 to 6.78+/-0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K-m values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) values for cellobiosides are higher for the mutant. This we attribute to reduced nonproductive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.

  • 6. Bessueille, Laurence
    et al.
    Sindt, Nicolas
    Guichardant, Michel
    Djerbi, Soraya
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Plasma membrane microdomains from hybrid aspen cells are involved in cell wall polysaccharide biosynthesis2009In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 420, 93-103 p.Article in journal (Refereed)
    Abstract [en]

    Detergent-resistant plasma membrane microdomains [DRMs (detergent-resistant membranes)] were isolated recently from several plant species. As for animal cells, a large range of cellular functions, such as signal transduction, endocytosis and protein trafficking, have been attributed to plant lipid rafts and DRMs. The data available are essentially based on protcomics and more approaches need to be undertaken to elucidate the precise function of individual populations of DRMs in plants. We report here the first isolation of DRMs from purified plasma membranes of a tree species, the hybrid aspen Populus tremula x tremuloides, and their biochemical characterization. Plasma membranes were solubilized with Triton X-100 and the resulting DRMs were isolated by flotation in sucrose density gradients. The DRMs were enriched in sterols, sphingolipids and glycosylphosphatidylinositol-anchored proteins and thus exhibited similar properties to DRMs from other species. However, they contained key carbohydrate synthases involved in cell wall polysaccharide biosynthesis, namely callose [(1 -> 3)-beta-D-glucan] and cellulose synthases. The association of these enzymes with DRMs was demonstrated using specific glucan synthase assays and antibodies, as well as biochemical and chemical approaches for the characterization of the polysaccharides synthesized in vitro by the isolated DRMs. More than 70% of the total glucan synthase activities present in the original plasma membranes was associated with the DRM fraction. In addition to shedding light on the lipid environment of callose and cellulose synthases, our results demonstrate the involvement of DRMs in the biosynthesis of important cell wall polysaccharides. This novel concept suggests a function of plant membrane microdomains in cell growth and morphogenesis.

  • 7.
    Blomqvist, Kristina
    et al.
    KTH, School of Biotechnology (BIO), Wood Biotechnology.
    Djerbi, Soraya
    KTH, School of Biotechnology (BIO), Wood Biotechnology.
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Wood Biotechnology.
    Teeri, Tuula
    KTH, School of Biotechnology (BIO), Wood Biotechnology.
    Cellulose Biosynthesis in Forest Trees2007In: Cellulose: Molecular and Structural Biology: Selected Articles on the Synthesis, Structure, and Applications of Cellulose / [ed] R. Malcolm Brown and Inder M. Saxena, Dordrecht: Springer Netherlands, 2007, 85-106 p.Chapter in book (Other academic)
    Abstract [en]

    Wood formation is a fundamental biological process of significant economic andcommercial interest. During wood formation, most glucose from the carbohydratemetabolism is channeled to cellulose in the secondary cell walls. The cellulose microfibrils associate with hemicellulose, proteins, and lignin to form the strong and flexiblebiocomposite known as wood. As the main wood component, cellulose is essential forthe survival of trees and for their exploitation by man.In spite of this, the molecular details of cellulose biosynthesis have remained obscure in all plants. In particular, the toughness of wood cells makes it hard to isolateactive enzymes and study cellulose synthesis in trees. Functional genomics providespowerful new tools to study complex metabolic processes. In this way, 18 CesA geneshave been recently identified in the genome sequence of Populus trichocarpa.Expression profiling during wood formation has shown that four of these genesare specifically upregulated during xylogenesis and/or tension wood formation. Othergenes that follow the same expression pattern as the wood-related CesA genes encodethe putative Korrigan ortholog PttCel9A and a novel microtubule associated proteinPttMAP20. Cell suspension cultures of hybrid aspen with elevated expression of thesecondary cell wall specific PttCesA genes have been used for efficient in vitro synthesisof cellulose, which will facilitate future studies of this challenging process in trees.

  • 8. Boer, H.
    et al.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Koivula, A.
    Characterization of Trichoderma reesei cellobiohydrolase CeI7A secreted from Pichia pastoris using two different promoters2000In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 69, no 5, 486-494 p.Article in journal (Refereed)
    Abstract [en]

    Heterologous expression of T. reesei cellobiohydrolase Cel7A in a methylotrophic yeast Pichia pastoris was tested both under the P. pastoris alcohol oxidase (AOX1) pro meter and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in a fermenter. Production of Ce17A with the AOX1 promoter gave a better yield, although part of the enzyme expressed was apparently not correctly folded. Cel7A expressed in P. pastoris is overglycosylated at its N-glycosylation sites as compared to the native T. reesei protein, but less extensive than Cel7A expressed in Saccharomyces cerevisiae. The k(cat) and K-m values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced. The measured pH optimum also closely resembles that of purified T. reesei Cel7A. Furthermore, the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements. On the other hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T. reesei Cel7A. The constitutive GAP promoter expression system of P. pastoris is nevertheless well suited for activity screening of cellulase activities in microtiter plates. With this type of screening method a faster selection of site-directed and random mutants with, for instance, an altered optimum pH is possible, in contrast to the homologous T. reesei expression system.

  • 9. Bollok, Monika
    et al.
    Henriksson, Hongbin
    Kallas, Åsa
    KTH, Superseded Departments, Biotechnology.
    Jahic, Mehmedalija
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tula
    KTH, Superseded Departments, Biotechnology.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Production of plant xyloglucan endotransglycosylase (XET) using the methylotrophic yeast Pichia pastorisIn: Applied Biochemistry and Biotechnology, ISSN 0273-2289, E-ISSN 1559-0291Article in journal (Other academic)
  • 10.
    Bollok, Monika
    et al.
    KTH, School of Biotechnology (BIO).
    Henriksson, Hongbin
    KTH, School of Biotechnology (BIO).
    Kallas, Åsa
    KTH, School of Biotechnology (BIO).
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast Pichia pastoris2005In: Applied Biochemistry and Biotechnology, ISSN 0273-2289, Vol. 126, 61-77 p.Article in journal (Refereed)
    Abstract [en]

    The gene XET16A encoding the enzyme xyloglucan endotransglycosylase (XET) from hybrid aspen (Populus tremula x tremuloides Mich) was transformed into Pichia pastoris GS115 and the enzyme was secreted to the medium. The influence of process conditions on the XET production, activity, and proteolytic degradation were examined. Inactivation of XET occurred in the foam, but could be decreased significantly by using an efficient antifoam. Rich medium (yeast extract plus peptone) was needed for product accumulation, but not for growth. The proteolytic degradation of the enzyme in the medium was substantially decreased by also adding yeast extract and peptone to the glycerol medium before induction with methanol. Decreasing the fermentation pH from 5.0 to 4.0 further reduced the proteolysis. The specific activity was further improved by production at 15 degrees C instead of 22 degrees C. In this way a XET production of 54 mg/L active enzyme could be achieved in the process with a specific activity of 18 Unit/mg protein after a downstream process including centrifugation, micro- and ultrafiltration, and ion exchange chromatography.

  • 11. Bourquin, V.
    et al.
    Nishikubo, N.
    Abe, H.
    Brumer, Harry
    KTH, Superseded Departments, Biotechnology.
    Denman, S.
    Eklund, M.
    Christiernin, M.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Sundberg, B.
    Mellerowicz, E. J.
    Xyloglucan endotransglycosylases have a function during the formation of secondary cell walls of vascular tissues2002In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 14, no 12, 3073-3088 p.Article in journal (Refereed)
    Abstract [en]

    Xyloglucan transglycosylases (XETs) have been implicated in many aspects of cell wall biosynthesis, but their function in vascular tissues, in general, and in the formation of secondary walls, in particular, is less well understood. Using an in situ XET activity assay in poplar stems, we have demonstrated XET activity in xylem and phloem fibers at the stage of secondary wall formation. Immunolocalization of fucosylated xylogucan with CCRC-M1 antibodies showed that levels of this species increased at the border between the primary and secondary wall layers at the time of secondary wall deposition. Furthermore, one of the most abundant XET isoforms in secondary vascular tissues (PttXET16A) was cloned and immunolocalized to fibers at the stage of secondary wall formation. Together, these data strongly suggest that XET has a previously unreported role in restructuring primary walls at the time when secondary wall layers are deposited, probably creating and reinforcing the connections between the primary and secondary wall layers. We also observed that xylogucan is incorporated at a high level in the inner layer of nacreous walls of mature sieve tube elements.

  • 12.
    Brumer, Harry
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Rutland, Mark
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Sinnott, M. L.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Cross-Linking Involving a Polymeric Carbohydrate Material2005Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    The present invention relates to a method of cross-linking a polymeric carbohydrate material with a second material by means of a soluble carbohydrate polymer and a crosslinking agent. The present invention furthermore relates to the resulting cross-linked material, to uses of the cross-linked material, as well as to a kit comprising the soluble carbohydrate polymer and the cross-linking agent.

  • 13.
    Brumer, Harry
    et al.
    KTH, Superseded Departments, Biotechnology.
    Zhou, Qi
    KTH, Superseded Departments, Biotechnology.
    Baumann, Martin J.
    KTH, Superseded Departments, Biotechnology.
    Carlsson, Kjell
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tuula
    KTH, Superseded Departments, Biotechnology.
    Activation of crystalline cellulose surfaces though the chemoenzymatic modification of xyloglucan2004In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, no 18, 5715-1721 p.Article in journal (Refereed)
    Abstract [en]

    Cellulose constitutes an important raw material for many industries. However, the superb load-bearing properties of cellulose are accompanied by poor chemical reactivity. The hydroxyl groups on cellulose surfaces can be reacted but usually not without loss of fiber integrity and strength. Here, we describe a novel chemoenzymatic approach for the efficient incorporation of chemical functionality onto cellulose surfaces. The modification is brought about by using a transglycosylating enzyme, xyloglucan endotranglycosylase, to join chemically modified xyloglucan oligosaccharides to xyloglucan, which has a naturally high affinity to cellulose. Binding of the chemically modified hemicellulose molecules can thus be used to attach a wide variety of chemical moieties without disruption of the individual fiber or fiber matrix.

  • 14. Christiernin, M.
    et al.
    Henriksson, Gunnar
    KTH, Superseded Departments, Pulp and Paper Technology.
    Lindström, Mikael
    KTH, Superseded Departments, Fibre and Polymer Technology.
    Brumer, Harry
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Lindström, T.
    Laine, J.
    The effects of xyloglucan on the properties of paper made from bleached kraft pulp2003In: Nordic Pulp & Paper Research Journal, ISSN 0283-2631, E-ISSN 2000-0669, Vol. 18, no 2, 182-187 p.Article in journal (Refereed)
    Abstract [en]

    Xyloglucan was adsorbed onto bleached soft-wood kraft pulp followed by preparation and analysis of handsheets with respect to sheet formation as well as sheet mechanical and optical properties. Adsorption of xyloglucan was found to be slow. After more than 20 hrs adsorption, equilibrium had not been reached. The amount of xyloglucan adsorbed increased with beating, but neither the rate of adsorption nor the quantity adsorbed was significantly affected by temperature. Xyloglucan was found to be practically irreversibly adsorbed onto the fibres and the effects of xyloglucan on paper sheet properties were investigated after thorough washing of the pulp. The adsorption characteristics of xyloglucan confirm observations by other authors on other cellulose substrates. Tensile index values for handsheets formed with the xyloglucan-containing pulps were higher than those measured for control pulps with a comparable beating degree. The light scattering coefficient was, however, not affected by xyloglucan adsorption. Hence, the increase in tensile strength is attributed to an increased relative bond strength between the fibres. Tensile strength versus tear strength relationship was similar for pulps with and without xyloglucan, but water retention value and dewatering resistance were lower for the xyloglucan treated pulps than for the reference pulps at the same tensile strength. In addition, formation was improved for pulps with adsorbed xyloglucan. The conclusion is that xyloglucan is a promising wet end additive that decreases the necessity for beating of the pulp and improves the formation of paper.

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

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

  • 16. Colombani, A.
    et al.
    Djerbi, Soraya
    KTH, Superseded Departments, Biotechnology.
    Bessueille, L.
    Blomqvist, Kristina
    KTH, Superseded Departments, Biotechnology.
    Ohlsson, Anna
    KTH, Superseded Departments, Biotechnology.
    Berglund, Torkel
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tuula
    KTH, Superseded Departments, Biotechnology.
    Bulone, V.
    In vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose by detergent extracts of membranes from cell suspension cultures of hybrid aspen2004In: Cellulose (London), ISSN 0969-0239, E-ISSN 1572-882X, Vol. 11, no 3-4, 313-327 p.Article in journal (Refereed)
    Abstract [en]

    The aim of this work was to optimize the conditions for in vitro synthesis of (1 --> 3)-beta-D-glucan (callose) and cellulose, using detergent extracts of membranes from hybrid aspen (Populus tremula x tremuloides) cells grown as suspension cultures. Callose was the only product synthesized when CHAPS extracts were used as a source of enzyme. The optimal reaction mixture for callose synthesis contained 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 8 mM Ca2+, and 20 mM cellobiose. The use of digitonin to extract the membrane-bound proteins was required for cellulose synthesis. Yields as high as 50% of the total in vitro products were obtained when cells were harvested in the stationary phase of the growth curve, callose being the other product. The optimal mixture for cellulose synthesis consisted of 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 1 mM Ca2+, 8 mM Mg2+, and 20 mM cellobiose. The in vitro beta-glucans were identified by hydrolysis of radioactive products, using specific enzymes. C-13-Nuclear magnetic resonance spectroscopy and transmission electron microscopy were also used for callose characterization. The (1-->3)-beta-D-glucan systematically had a microfibrillar morphology, but the size and organization of the microfibrils were affected by the nature of the detergent used for enzyme extraction. The discussion of the results is included in a short review of the field that also compares the data obtained with those available in the literature. The results presented show that the hybrid aspen is a promising model for in vitro studies on callose and cellulose synthesis.

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

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

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

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

  • 19.
    Djerbi, Soraya
    et al.
    KTH, Superseded Departments, Biotechnology.
    Aspeborg, Henrik
    KTH, Superseded Departments, Biotechnology.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    Blomqvist, Kristina
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tuula
    KTH, Superseded Departments, Biotechnology.
    Identification and expression analysis of genes encoding putative cellulose synthases (CesA) in the hybrid aspen, Populus tremula (L.) × P. tremuloides (Michx.)2004In: Cellulose (London), ISSN 0969-0239, E-ISSN 1572-882X, Vol. 11, no 3-4, 301-312 p.Article in journal (Refereed)
    Abstract [en]

    Cellulose is synthesized in plant cell walls by large membrane-bound protein complexes proposed to contain several copies of the catalytic subunit of the cellulose synthase, CesA. Here we report identification of 10 distinct CesA genes within a database of 100,000 ESTs of the hybrid aspen, Populus tremula (L.) x P. tremuloides (Michx.). Expression analyses in normal wood undergoing xylogenesis and in tension wood indicate xylem specific expression of four putative CesA isoenzymes, PttCesA1, PttCesA3-1, PttCesA3-2 and PttCesA9. Both the protein sequences and the expression profiles of PttCesA3-1 and PttCesA3-2 are very similar, and they may thus represent redundant copies of an enzyme with essentially the same function. Further, one of the generally more constitutively expressed CesA genes, PttCesA2, seems to be activated on the opposite side of a tension wood induced stem, while PttCesA6 appears to be more specific for leaf tissues. The rest of the hybrid aspen CesA genes were found to be relatively evenly expressed over the poplar tissues hereby studied.

  • 20.
    Djerbi, Soraya
    et al.
    KTH, School of Biotechnology (BIO).
    Lindskog, Mats
    KTH, School of Biotechnology (BIO).
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Numerical Analysis and Computer Science, NADA.
    Sterky, Fredrik
    KTH, School of Biotechnology (BIO).
    Teeri, Tuula
    KTH, School of Biotechnology (BIO).
    The genome sequence of black cottonwood (Populus trichocarpa) reveals 18 conserved cellulose synthase (CesA) genes2005In: Planta, ISSN 0032-0935, E-ISSN 1432-2048, Vol. 221, no 5, 739-746 p.Article in journal (Refereed)
    Abstract [en]

    The genome sequence of Populus trichocarpa was screened for genes encoding cellulose synthases by using full-length cDNA sequences and ESTs previously identified in the tissue specific cDNA libraries of other poplars. The data obtained revealed 18 distinct CesA gene sequences in P. trichocarpa. The identified genes were grouped in seven gene pairs, one group of three sequences and one single gene. Evidence from gene expression studies of hybrid aspen suggests that both copies of at least one pair, CesA3-1 and CesA3-2, are actively transcribed. No sequences corresponding to the gene pair, CesA6-1 and CesA6-2, were found in Arabidopsis or hybrid aspen, while one homologous gene has been identified in the rice genome and an active transcript in Populus tremuloides. A phylogenetic analysis suggests that the CesA genes previously associated with secondary cell wall synthesis originate from a single ancestor gene and group in three distinct subgroups. The newly identified copies of CesA genes in P. trichocarpa give rise to a number of new questions concerning the mechanism of cellulose synthesis in trees.

  • 21.
    Eklund, Malin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Sandström, Kristofer
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tuula
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Site-specific and reversible anchoring of active proteins onto cellulose using a cellulosome-like complex2004In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 109, no 3, 277-286 p.Article in journal (Refereed)
    Abstract [en]

    Protein engineering strategies facilitating controlled and spontaneous assembly of macromolecular complexes are of great interest for the design of artificial multi-enzyme systems of pre-defined composition. Here we have combined affinity proteins from different sources to achieve specific and reversible anchoring of affinity domain-tagged reporter proteins to a cell ulose-anchored fusion protein. The design principle mimics the architecture of macromolecular cellulosome complexes produced by some cellulolytic microbes. A fusion protein between a cellulose-binding module (CBM1(Cel6A)) of the Trichoderma reesei cellobiohydrolase Cel6A and a five-domain staphylococcal protein A (SPA) was constructed to serve as platform for docking of easily detectable reporter proteins onto cellulose surfaces. In turn, the reporter proteins were produced as fusions to two copies of a SPA-binding affinity protein (an affibody denoted Z(SPA-1)), selected from a phage display library constructed by combinatorial protein engineering. In a series of experiments, involving repeated washing and low pH elution, affinity-tagged Enhanced Green Fluorescent Protein (EGFP) and Fusarium solani pisi lipase cutinase reporter proteins were both found to be specifically directed from solution to the same region of a cellulose filter paper where SPA-CBM1(Cel6A) fusion protein had been previously applied. This showed that the SPA-CBM1(Cel6A) fusion protein had been stably anchored to the cellulose surface without loss of binding capacity and that the interaction between SPA and the Z(SPA-1) affibody domains was selective. The generality of this biospecificity-driven system for assembly applications is discussed.

  • 22. Filonova, Lada
    et al.
    Kallas, Åsa M.
    KTH, School of Biotechnology (BIO).
    Greffe, Lionel
    KTH, School of Biotechnology (BIO).
    Teeri, Tuula T.
    Daniel, Geoffrey
    Johansson, Gunnar
    Mapping of crystalline cellulose and mannan on the surfaces of wood tissues and pulp fibers using carbohydrate binding modules2007In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 8, no 1, 91-97 p.Article in journal (Refereed)
    Abstract [en]

    Carbohydrate binding modules (CBMs) are noncatalytic substrate binding domains of many enzymes involved in carbohydrate metabolism. Here we used fluorescent labeled recombinant CBMs specific for crystalline cellulose (CBM1(HjCel7A)) and mannans (CBM27(TmMan5) and CBM35(CjMan5C)) to analyze the complex surfaces of wood tissues and pulp fibers. The crystalline cellulose CBM1(HjCel7A) was found as a reliable marker of both bacterially produced and plant G-layer cellulose, and labeling of spruce pulp fibers with CBM1(HjCel7A) revealed a signal that increased with degree of fiber damage. The mannan-specific CBM27(TmMan5) and CBM35(CjMan5C) CBMs were found to be more specific reagents than a monoclonal antibody specific for (1 -> 4)-beta-mannan/galacto-(1 -> 4)-beta-mannan for mapping carbohydrates on native substrates. We have developed a quantitative fluorometric method for analysis of crystalline cellulose accumulation on fiber surfaces and shown a quantitative difference in crystalline cellulose binding sites in differently processed pulp fibers. Our results indicated that CBMs provide useful, novel tools for monitoring changes in carbohydrate content of nonuniform substrate surfaces, for example, during wood or pulping processes and possibly fiber biosynthesis.

  • 23.
    Fugelstad, Johanna
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bouzenzana, Jamel
    Djerbi, Soraya
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Numerical Analysis and Computer Science, NADA.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    A novel family of cellulose synthase genes from the Oomycete Saprolegnia monoica: functional characterization using cellulose synthesis inhibitorsManuscript (Other academic)
  • 24.
    Fugelstad, Johanna
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bouzenzana, Jamel
    Djerbi, Soraya
    Guerriero, Gea
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Identification of the cellulose synthase genes from the Oomycete Saprolegnia monoica and effect of cellulose synthesis inhibitors on gene expression and enzyme activity2009In: Fungal Genetics and Biology, ISSN 1087-1845, E-ISSN 1096-0937, Vol. 46, no 10, 759-767 p.Article in journal (Refereed)
    Abstract [en]

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

  • 25. Geisler-Lee, J.
    et al.
    Geisler, M.
    Coutinho, P. M.
    Segerman, B.
    Nishikubo, N.
    Takahashi, J.
    Aspeborg, H.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Djerbi, S.
    Master, E.
    Andersson-Gunneras, S.
    Sundberg, B.
    Karpinski, S.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kleczkowski, L. A.
    Henrissat, B.
    Mellerowicz, E. J.
    Poplar carbohydrate-active enzymes. Gene identification and expression analyses2006In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 140, no 3, 946-962 p.Article in journal (Refereed)
    Abstract [en]

    Over 1,600 genes encoding carbohydrate-active enzymes (CAZymes) in the Populus trichocarpa (Torr.&Gray) genome were identified based on sequence homology, annotated, and grouped into families of glycosyltransferases, glycoside hydrolases, carbohydrate esterases, polysaccharide lyases, and expansins. Poplar ( Populus spp.) had approximately 1.6 times more CAZyme genes than Arabidopsis ( Arabidopsis thaliana). Whereas most families were proportionally increased, xylan and pectin-related families were underrepresented and the GT1 family of secondary metabolite-glycosylating enzymes was overrepresented in poplar. CAZyme gene expression in poplar was analyzed using a collection of 100,000 expressed sequence tags from 17 different tissues and compared to microarray data for poplar and Arabidopsis. Expression of genes involved in pectin and hemicellulose metabolism was detected in all tissues, indicating a constant maintenance of transcripts encoding enzymes remodeling the cell wall matrix. The most abundant transcripts encoded sucrose synthases that were specifically expressed in wood-forming tissues along with cellulose synthase and homologs of KORRIGAN and ELP1. Woody tissues were the richest source of various other CAZyme transcripts, demonstrating the importance of this group of enzymes for xylogenesis. In contrast, there was little expression of genes related to starch metabolism during wood formation, consistent with the preferential flux of carbon to cell wall biosynthesis. Seasonally dormant meristems of poplar showed a high prevalence of transcripts related to starch metabolism and surprisingly retained transcripts of some cell wall synthesis enzymes. The data showed profound changes in CAZyme transcriptomes in different poplar tissues and pointed to some key differences in CAZyme genes and their regulation between herbaceous and woody plants.

  • 26. Gray-Mitsumune, Madoka
    et al.
    Blomquist, Kristina
    McQueen-Mason, Simon
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Sundberg, Bjoern
    Mellerowicz, Ewa J.
    Ectopic expression of a wood-abundant expansin PttEXPA1 promotes cell expansion in primary and secondary tissues in aspen2008In: Plant Biotechnology Journal, ISSN 1467-7644, E-ISSN 1467-7652, Vol. 6, no 1, 62-72 p.Article in journal (Refereed)
    Abstract [en]

    Expansins are primary agents inducing cell wall extension, and are therefore obvious targets in biotechnological applications aimed at the modification of cell size in plants. In trees, increased fibre length is a goal of both breeding and genetic engineering programmes. We used an alpha-expansin PttEXPA1 that is highly abundant in the wood-forming tissues of hybrid aspen (Populus tremula L. x P. tremuloides Michx.) to evaluate its role in fibre elongation and wood cell development. PttEXPA1 belongs to Subfamily A of alpha-expansins that have conserved motifs at the N- and C-termini of the mature protein. When PttEXPA1 was over-expressed in aspen, an extract of the cell wall-bound proteins of the transgenic plants exhibited an increased expansin activity on cellulose-xyloglucan composites in vitro, indicating that PttEXPA1 is an active expansin. The transgenic lines exhibited increased stem internode elongation and leaf expansion, and larger cell sizes in the leaf epidermis, indicating that PttEXPA1 protein is capable of increasing the growth of these organs by enhancing cell wall expansion in planta. Wood cell development was also modified in the transgenic lines, but the effects were different for vessel elements and fibres, the two main cell types of aspen wood. PttEXPA1 stimulated fibre, but not vessel element, diameter growth, and marginally increased vessel element length, but did not affect fibre length. The observed differences in responsiveness to expansin of these cell types are discussed in the light of differences in their growth strategies and cell wall composition.

  • 27. Gray-Mitsumune, Madoka
    et al.
    Mellerowicz, Ewa J.
    Abe, Hisashi
    Schrader, Jarmo
    Winzell, Anders
    KTH, Superseded Departments, Biotechnology.
    Sterky, Fredrik
    KTH, Superseded Departments, Biotechnology.
    Blomqvist, Kristina
    KTH, Superseded Departments, Biotechnology.
    McQueen-Mason, Simon
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Sundberg, Björn
    Expansins abundant in secondary xylem belong to subgroup a of the alpha-expansin gene family (1 w )2004In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 135, no 3, 1552-1564 p.Article in journal (Refereed)
    Abstract [en]

    Differentiation of xylem cells in dicotyledonous plants involves expansion of the radial primary cell walls and intrusive tip growth of cambial derivative cells prior to the deposition of a thick secondary wall essential for xylem function. Expansins are cell wall-residing proteins that have an ability to plasticize the cellulose-hemicellulose network of primary walls. We found expansin activity in proteins extracted from the cambial region of mature stems in a model tree species hybrid aspen (Populus tremula X Populus tremuloides Michx). We identified three a-expansin genes (PttEXP1, PttEXP2, and PttEXP8) and one beta-expansin gene (PttEXPB1) in a cambial region expressed sequence tag library, among which PttEXP1 was most abundantly represented. Northern-blot analyses in aspen vegetative organs and tissues showed that PttEXP1 was specifically expressed in mature stems exhibiting secondary growth, where it was present in the cambium and in the radial expansion zone. By contrast, PttEXP2 was mostly expressed in developing leaves. In situ reverse transcription-PCR provided evidence for accumulation of mRNA of PttEXP1 along with ribosomal rRNA at the tips of intrusively growing xylem fibers, suggesting that PttEXP1 protein has a role in intrusive tip growth. An examination of tension wood and leaf cDNA libraries identified another expansin, PttEXP5, very similar to PttEXP1, as the major expansin in developing tension wood, while PttEXP3 was the major expansin expressed in developing leaves. Comparative analysis of expansins expressed in woody stems in aspen, Arabidopsis, and pine showed that the most abundantly expressed expansins share sequence similarities, belonging to the subfamily A of alpha-expansins and having two conserved motifs at the beginning and end of the mature protein, RIPVG and KNFRV, respectively. This conservation suggests that these genes may share a specialized, not yet identified function.

  • 28.
    Gullfot, Fredrika
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Teeri, Tuula
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Design of GH16 XET/XEH chimeric enzymes with SCHEMA: ManuscriptManuscript (preprint) (Other academic)
    Abstract [en]

    This manuscript contains experimental data obtained during a pilot study for the application of the SCHEMA method for structure-guided recombination on PttXET16-34 and TmNXG1, a model system for the evolution of different catalytic routes of GH16 XETs and XEHs.

    A restricted library of PttXET16-34/TmNXG1 chimeras with high diversity and low calculated SCHEMA disruption was generated based on crossover points identified by the RASPP algorithm. Analysis of the library revealed a bias among certain regions to remain intact and recalcitrant to recombination, in particular the upper and lower β-sheet structures forming the part of the protein that binds the donor substrate. In contrast, sequence diversity was preferentially introduced at the N-terminus, the major part of the acceptor side of the protein, and most of the C-terminal extension characteristic to XET/XEH in the GH16 family. Finally, in order to test the predictive capacity of SCHEMA, six chimeras with low calculated disruption were chosen for subsequent cloning and expression in Pichia pastoris.

  • 29. Gustavsson, M.
    et al.
    Lehtio, J.
    Denman, S.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Martinelle, Mats
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris2001In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 14, no 9, 711-715 p.Article in journal (Refereed)
    Abstract [en]

    Fusion proteins composed of a cellulose-binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B were constructed using different linker peptides. The aim was to create proteolytically stable linkers that were able to join the functional modules without disrupting their function. Six fusion variants containing linkers of 4-44 residues were expressed in Pichia pastoris and analysed. Three variants were found to be stable throughout 7-day cultivations. The cellulose-binding capacities of fusion proteins containing short linkers were slightly lower compared with those containing long linkers. The lipase-specific activities of all variants, in solution or immobilized on to cellulose, were equal to that of the wildtype lipase.

  • 30. Gustavsson, M. T.
    et al.
    Persson, P. V.
    Iversen, T.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Modification of cellulose fiber surfaces by use of a lipase and a xyloglucan endotransglycosylase2005In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 6, no 1, 196-203 p.Article in journal (Refereed)
    Abstract [en]

    A strategy for the modification of cellulose fiber surfaces was developed that used the ability of Candida antarctica lipase B (CALB) to acylate carbohydrates with high regioselectivity, combined with the transglycosylating activity of the Populus tremula x P. tremuloides xyloglucan endotransglycosylase 16A (PttXET16A). Xyloglucan oligosaccharides (XGOs) prepared from tamarind xyloglucan were acylated with CALB as a catalyst and vinyl stearate or gamma-thiobutyrolactone as acyl donors to produce carbohydrate molecules with hydrophobic alkyl chains or reactive sulfhydryl groups, respectively. The modified XGOs were shown to act as glycosyl acceptors in the transglycosylation reaction catalyzed by PttXET16A and could therefore be incorporated into high M-r xyloglucan chains. The resulting xyloglucan molecules exhibited a high affinity for cellulose surfaces, which enabled the essentially irreversible introduction of fatty acid esters or thiol groups to cellulose fibers.

  • 31.
    Henriksson, Gunnar
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Teeri, Tuula
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Biotechnology in the forest industry2009In: Pulp and Paper Chemistry and Technology: Wood Chemistry and Biotechnology / [ed] Ek, Monica / Gellerstedt, Göran / Henriksson, Gunnar, Berlin: Walter de Gruyter, 2009, 273-300 p.Chapter in book (Refereed)
  • 32. Henriksson, H.
    et al.
    Denman, S. E.
    Campuzano, I. D. G.
    Ademark, P.
    Master, E. R.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Brumer, Harry
    KTH, Superseded Departments, Biotechnology.
    N-linked glycosylation of native and recombinant cauliflower xyloglucan endotransglycosylase 16A2003In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 375, 61-73 p.Article in journal (Refereed)
    Abstract [en]

    The gene encoding a XET (xyloglucan endotransglycosylase) from cauliflower (Brassica oleracea var. botrytis) florets has been cloned and sequenced. Sequence analysis indicated a high degree of similarity to other XET enzymes belonging to glycosyl hydrolase family 16 (GH16). In addition to the conserved GH16 catalytic sequence motif EIDFE, there exists one potential N-linked glycosylation site. which is also highly conserved in XET enzymes from this family. Purification of the corresponding protein from extracts of cauliflower florets allowed the fractionation of a single, pure glycoform. which was analysed by MS techniques. Accurate protein mass determination following the enzymic deglycosylation of this glycoform indicated the presence of a high-mannose-type glycan of the general structure GlcNAc(2)Man(6). LC/MS and MS/MS (tandem MS) analysis provided supporting evidence for this structure and confirmed that the glycosylation site (underlined) was situated close to the predicted catalytic residues in the conserved sequence YLSSTNNEHDEIDFEFLGNRTGQPVILQTNVFTGGK. Heterologous expression in Pichia pastoris produced a range of protein glycoforms, which were, on average, more highly mannosylated than the purified native enzyme. This difference in glycosylation did not influence the apparent enzymic activity of the enzyme significantly. However, the removal of high-mannose glycosylation in recombinant cauliflower XET by endoglycosidase H, quantified by electrospray-ionization MS, caused a 40 % decrease in the transglycosylation activity of the enzyme. No hydrolytic activity was detected in native or heterologously expressed BobXET16A, even when almost completely deglycosylated.

  • 33.
    Henriksson, Maria
    et al.
    KTH, School of Biotechnology (BIO).
    Teeri, Tuula
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Improvements of Pichia pastoris fermentation technique for production of recombinant proteinsManuscript (Other academic)
  • 34. Hertzberg, M.
    et al.
    Aspeborg, H.
    Schrader, J.
    Andersson, A.
    Erlandsson, R.
    Blomqvist, K.
    Bhalerao, R.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Sundberg, B.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    Sandberg, G.
    A transcriptional roadmap to wood formation2001In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 98, no 25, 14732-14737 p.Article in journal (Refereed)
    Abstract [en]

    The large vascular meristem of poplar trees with its highly organized secondary xylem enables the boundaries between different developmental zones to be easily distinguished. This property of wood-forming tissues allowed us to determine a unique tissue-specific transcript profile for a well defined developmental gradient. RNA was prepared from different developmental stages of xylogenesis for DNA microarray analysis by using a hybrid aspen unigene set consisting of 2,995 expressed sequence tags. The analysis revealed that the genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of transcription factors and other potential regulators of xylogenesis, are under strict developmental stage-specific transcriptional regulation.

  • 35. Hrmova, Maria
    et al.
    Farkas, Vladimir
    Harvey, Andrew J.
    Lahnstein, Jelle
    Wischmann, Bente
    Kaewthai, Nomchit
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fincher, Geoffrey B.
    Substrate specificity and catalytic mechanism of a xyloglucan xyloglucosyl transferase HvXET6 from barley (Hordeum vulgare L.)2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 2, 437-456 p.Article in journal (Refereed)
    Abstract [en]

    A family 16 glycoside hydrolase, xyloglucan xyloglucosyl transferase (EC 2.4.1.207), also known as xyloglucan endotransglycosylase (XET), and designated isoenzyme HvXET6, was purified approximately 400-fold from extracts of young barley seedlings. The complete amino acid sequence of HvXET6 was deduced from the nucleotide sequence of a near full-length cDNA, in combination with tryptic peptide mapping. An additional five to six isoforms or post-translationally modified XET enzymes were detected in crude seedling extracts of barley. The HvXET6 isoenzyme was expressed in Pichia pastoris, characterized and compared with the previously purified native HvXET5 isoform. Barley HvXET6 has a similar apparent molecular mass of 33-35 kDa to the previously purified HvXET5 isoenzyme, but the two isoenzymes differ in their isoelectric points, pH optima, kinetic properties and substrate specificities. The HvXET6 isoenzyme catalyses transfer reactions between xyloglucans and soluble cellulosic substrates, using oligo-xyloglucosides as acceptors, but at rates that are significantly different from those observed for HvXET5. No hydrolytic activity could be detected with either isoenzyme. Comparisons of the reaction rates using xyloglucan or hydroxyethyl cellulose as donors and a series of cellodextrins as acceptors indicated that the acceptor site of HvXET can accommodate five glucosyl residues. Molecular modelling supported this conclusion and further confirmed the ability of the enzyme's active site to accommodate xyloglucan and cellulosic substrates. The two HvXETs followed a ping-pong (Bi, Bi) rather than a sequential reaction mechanism.

  • 36. Jahic, Mehmedalija
    et al.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Charoenrat, Theppanya
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Process technology for production and recovery of heterologous proteins with Pichia pastoris2006In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 22, no 6, 1465-1473 p.Article, review/survey (Refereed)
    Abstract [en]

    Developments in process techniques for production and recovery of heterologous proteins with Pichia pastoris are presented. Limitations for the standard techniques are described, and alternative techniques that solve the limitations problems are reviewed together with the methods that resulted in higher productivity of the P. pastoris processes. The main limitations are proteolysis of the secreted products and cell death in the high cell density bioreactor cultures. As a consequence, both low productivity and lower quality of the feedstock for downstream processing are achieved in processes hampered with these problems. Methods for exploring proteolysis and cell death are also presented. Solving the problems makes the conditions for downstream processing superior for the P. pastoris expression systems compared to other systems, which either need complex media or rely on intracellular production. These improved conditions allow for interfacing of cultivation with downstream processing in an integrated fashion.

  • 37. Johansson, P.
    et al.
    Brumer, Harry
    KTH, Superseded Departments, Biotechnology.
    Baumann, Martin J.
    KTH, Superseded Departments, Biotechnology.
    Kallas, Åsa
    KTH, Superseded Departments, Biotechnology.
    Henriksson, Hongbin
    KTH, Superseded Departments, Biotechnology.
    Denman, Stuart
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tuula
    KTH, Superseded Departments, Biotechnology.
    Jones, A.
    Crystal structures of a poplar xyloglucan endotransglycosylase reveal details of the transglycosylation acceptor binding2004In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 16, no 4, 874-886 p.Article in journal (Refereed)
    Abstract [en]

    Xyloglucan endotransglycosylases (XETs) cleave and religate xyloglucan polymers in plant cell walls via a transglycosylation mechanism. Thus, XET is a key enzyme in all plant processes that require cell wall remodeling. To provide a basis for detailed structure-function studies, the crystal structure of Populus tremula x tremuloides XET16A (PttXET16A), heterologously expressed in Pichia pastoris, has been determined at 1.8-Angstrom resolution. Even though the overall structure of PttXET16A is a curved beta-sandwich similar to other enzymes in the glycoside hydrolase family GH16, parts of its substrate binding cleft are more reminiscent of the distantly related family GH7. In addition, XET has a C-terminal extension that packs against the conserved core, providing an additional beta-strand and a short alpha-helix. The structure of XET in complex with a xyloglucan nonasaccharide, XLLG, reveals a very favorable acceptor binding site, which is a necessary but not sufficient prerequisite for transgilycosylation. Biochemical data imply that the enzyme requires sugar residues in both acceptor and donor sites to properly orient the glycosidic bond relative to the catalytic residues.

  • 38. Johansson, P.
    et al.
    Denman, S.
    Brumer, Harry
    KTH, Superseded Departments, Biotechnology.
    Kallas, A. M.
    Henriksson, H.
    Bergfors, T.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Jones, T. A.
    Crystallization and preliminary X-ray analysis of a xyloglucan endotransglycosylase from Populus tremula x tremuloides2003In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 59, 535-537 p.Article in journal (Refereed)
    Abstract [en]

    Xyloglucan endotransglycosylases (XETs) cleave and religate xyloglucan polymers in plant cell walls. Recombinant XET from poplar has been purified from a Pichia pastoris expression system and crystallized. Two different crystal forms were obtained by vapour diffusion from potassium sodium tartrate and from an imidazole buffer using sodium acetate as a precipitant. Data were collected from these crystal forms to 3.5 and 2.1 Angstrom resolution, respectively. The first crystal form was found to belong to space group P3(1)21 or P3(2)21 (unit-cell parameters a = 98.6, b = 98.6, c = 98.5 Angstrom) and the second crystal form to space group P6(3) (unit-cell parameters a = 188.7, b = 188.7, c = 46.1 Angstrom).

  • 39.
    Jonsson Rudsander, Ulla
    et al.
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Sandstrom, Corine
    Piens, Kathleen
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Master, Emma
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Wilson, David B.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime. KTH, School of Biotechnology (BIO), Glycoscience.
    Kenne, Lennart
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime. KTH, School of Biotechnology (BIO), Glycoscience.
    Comparative NMR analysis of cellooligosaccharide hydrolysis by GH9 bacterial and plant endo-1,4-ss-glucanases2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 18, 5235-5241 p.Article in journal (Refereed)
    Abstract [en]

    H-1 NMR spectroscopy has been used to analyze the product profiles arising from the hydrolysis of cellooligosaccharides by family GH9 cellulases. The product profiles obtained with the wild type and several active site mutants of a bacterial processive endoglucanase, Tf Cel9A, were compared with those obtained by a randomly acting plant endoglucanase, PttCe19A. PttCe19A is an orthologue of the Arabidopsis endocellulase, Korrigan, which is required for efficient cellulose biosynthesis. As expected, poplar PttCe19A was shown to catalyze the degradation of cellooligosaccharides by inversion of the configuration of the anomeric carbon. The product analyses showed that the number of interactions between the glucose units of the substrate and the aromatic residues in the enzyme active sites determines the point of cleavage in both enzymes.

  • 40.
    Kaewthai, Nomchit
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Harvey, Andrew J.
    Hrmova, Maria
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fincher, Geoffrey B.
    Heterologous expression of diverse barley XTH genes in the yeast Pichia pastoris2010In: PLANT BIOTECHNOLOGY, ISSN 1342-4580, Vol. 27, no 3, 251-258 p.Article in journal (Refereed)
    Abstract [en]

    Heterologous expression of plant genes, particularly those encoding carbohydrate-active enzymes such as glycoside hydrolases and glycosyl transferases, continues to be a major hurdle in the functional analysis of plant proteomes. Presently, there are few convenient systems for the production of recombinant plant enzymes in active form and at adequate levels for biochemical and structural characterization. The methylotrophic yeast Pichia pastoris is an attractive expression host due to its ease of manipulation and its capacity to perform post-translational protein modifications, such as N-glycosylation [Daly and Hearn (2005) J Mol Recognit 18: 119-138]. Here, we demonstrate the utility of the P. pastoris SMD1168H/pPICZ-alpha C system for the expression of a range of xyloglucan endo-transglycosylase/hydrolase (XTH) cDNAs from barley (Hordeum vulgare). Although stable transformants were readily obtained by positive selection for vector-induced antibiotic resistance for all of the nine constructs tested, only five isoforms were secreted as soluble proteins into the culture medium, four in active form. Furthermore, production levels of these five isoforms were found to be variable, depending on the transformant, which further underscores the necessity of screening multiple clones for expression of active enzyme. Failure to express certain XTH isoforms in P. pastoris could not be correlated with any conserved gene or protein sequence properties, and this precluded using rational sequence engineering to enhance heterologous expression of the cDNAs. Thus, while significant advances are reported here, systems for the heterologous production of plant proteins require further development.

  • 41.
    Kallas, Åsa M.
    et al.
    KTH, School of Biotechnology (BIO).
    Baumann, Martin J.
    KTH, School of Biotechnology (BIO).
    Fäldt, Jenny
    KTH.
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO).
    Denman, Stuart
    KTH.
    Mellerowicz, Ewa J.
    Nishikubo, Nobuyushi
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO).
    Enzymatic characterization of a recombinant xyloglucan endotransglycosylase PttXET16-35 from Populus tremula x tremuloidesManuscript (Other academic)
  • 42.
    Kallas, Åsa M.
    et al.
    KTH, School of Biotechnology (BIO).
    Coutinho, Pedro
    Bulone, Vincent
    KTH, School of Biotechnology (BIO).
    Mellerowicz, Ewa J.
    Gilbert, Harry J.
    Henrissat, Bernard
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO).
    Characterization of a CBM43 module from hybrid aspen (Populus tremula x tremuloides): specificity of polysaccharide interaction and bioinformatic analysisManuscript (Other academic)
  • 43.
    Kallas, Åsa M.
    et al.
    KTH, School of Biotechnology (BIO).
    Piens, Kathleen
    KTH, School of Biotechnology (BIO).
    Denman, Stuart E.
    KTH, School of Biotechnology (BIO).
    Henriksson, Hongbin
    KTH, School of Biotechnology (BIO).
    Fäldt, Jenny
    KTH, School of Chemical Science and Engineering (CHE).
    Johansson, Patrik
    Brumer, Harry
    KTH, School of Biotechnology (BIO).
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO).
    Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula x tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris2005In: Biochemical Journal, ISSN 0264-6021, Vol. 390, 105-113 p.Article in journal (Refereed)
    Abstract [en]

    The cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (Populus tremula x tremuloides) has been isolated from an expressed sequence tag library and expressed in the methylotrophic yeast Pichia pastoris. Sequence analysis indicated a high degree of similarity with other proteins in the XTH (xyloglucan transglycosylase/hydrolase) gene subfamily of GH16 (glycoside hydrolase family 16). In addition to the conserved GH16 catalytic sequence motif, PttXET16A contains a conserved N-glycosylation site situated proximal to the predicted catalytic residues. MS analysis indicated that the recombinant PttXET16A expressed in P. pastoris is heterogeneous due to the presence of variable N-glycosylation and incomplete cleavage of the a-factor secretion signal peptide. Removal of the N-glycan by endoglycosidase H treatment did not influence the catalytic activity significantly. Similarly, site-directed mutagenesis of Asn(93) to serine to remove the N-glycosylation site resulted in an enzyme which was comparable with the wild-type enzyme in specific activity and thermal stability but had clearly reduced solubility. Hydrolytic activity was detected neither in wild-type PttXET16A before or after enzymatic deglycosylation nor in PttXET16A N93S (Asn(93) -> Ser) mutant.

  • 44. Koivula, A.
    et al.
    Ruohonen, L.
    Wohlfahrt, G.
    Reinikainen, T.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Piens, K.
    Claeyssens, M.
    Weber, M.
    Vasella, A.
    Becker, D.
    Sinnott, M. L.
    Zou, J. Y.
    Kleywegt, G. J.
    Szardenings, M.
    Stahlberg, J.
    Jones, T. A.
    The active site of cellobiohydrolase Cel6A from Trichoderma reesei: The roles of aspartic acids D221 and D1752002In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 124, no 34, 10015-10024 p.Article in journal (Refereed)
    Abstract [en]

    Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other. Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve a catalytic base. The value of (D2O)(V) of 1.16 +/- 0.14 for hydrolysis of cellotriose suggests that the large direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond between D221 and D175 before catalysis.

  • 45. Kumar, Manoj
    et al.
    Thammannagowda, Shivegowda
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Wood Biotechnology.
    Chiang, Vincent
    Han, Kyung-Hwan
    Joshi, Chandrashekhar P.
    Mansfield, Shawn D.
    Mellerowicz, Ewa
    Sundberg, Bjorn
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Wood Biotechnology.
    Ellis, Brian E.
    An update on the nomenclature for the cellulose synthase genes in Populus2009In: Trends in Plant Science, ISSN 1360-1385, E-ISSN 1878-4372, Vol. 14, no 5, 248-254 p.Article, review/survey (Refereed)
    Abstract [en]

    Cellulose synthase (CesA) is a central catalyst in the generation of the plant cell wall biomass and is, therefore, the focus of intense research. Characterization of individual CesA genes from Populus species has led to the publication of several different naming conventions for CesA gene family members in this model tree. To help reduce the resulting confusion, we propose here a new phylogeny-based CesA nomenclature that aligns the Populus CesA gene family with the established Arabidopsis thaliana CesA family structure.

  • 46. Lehtio, J.
    et al.
    Sugiyama, J.
    Gustavsson, M.
    Fransson, L.
    Linder, M.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    The binding specificity and affinity determinants of family 1 and family 3 cellulose binding modules2003In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 100, no 2, 484-489 p.Article in journal (Refereed)
    Abstract [en]

    Cellulose binding modules (CBMs) potentiate the action of cellulolytic enzymes on insoluble substrates. Numerous studies have established that three aromatic residues on a CBM surface are needed for binding onto cellulose crystals and that tryptophans contribute to higher binding affinity than tyrosines. However, studies addressing the nature of CBM-cellulose interactions have so far failed to establish the binding site on cellulose crystals targeted by CBMs. In this study, the binding sites of CBMs on Valonia cellulose crystals have been visualized by transmission electron microscopy. Fusion of the CBMs with a modified staphylococcal protein A (ZZ-domain) allowed direct immuno-gold labeling at close proximity of the actual CBM binding site. The transmission electron microscopy images provide unequivocal evidence that the fungal family 1 CBMs as well as the family 3 CBM from Clostridium thermocellum CipA have defined binding sites on two opposite corners of Valonia cellulose crystals. In most samples these corners are worn to display significant area of the hydrophobic (110) plane, which thus constitutes the binding site for these CBMs.

  • 47. Lehtio, J.
    et al.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Alpha-amylase inhibitors selected from a combinatorial library of a cellulose binding domain scaffold2000In: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 41, no 3, 316-322 p.Article in journal (Refereed)
    Abstract [en]

    A disulfide bridge-constrained cellulose binding domain (CBD,) derived from the cellobiohydrolase Ce17A from Trichoderma reesei has been investigated for use in scaffold engineering to obtain novel binding proteins. The gene encoding the wild-type 36 aa CBDWT domain was first inserted into a phagemid vector and shown to be functionally displayed on M13 filamentous phage as a protein III fusion protein with retained cellulose binding activity. A combinatorial library comprising 46 million variants of the CBD domain was constructed through randomization of 11 positions located at the domain surface and distributed over three separate beta -sheets of the domain. Using the enzyme porcine alpha-amylase (PPA) as target in biopannings, two CBD variants showing selective binding to the enzyme were characterized. Reduction and iodoacetamide blocking of cysteine residues in selected CBD variants resulted in a loss of binding activity, indicating a conformation dependent binding. Interestingly, further studies showed that the selected CBD variants were capable of competing with the binding of the amylase inhibitor acarbose to the enzyme. In addition, the enzyme activity could be partially inhibited by addition of soluble protein, suggesting that the selected CBD variants bind to the active site of the enzyme.

  • 48. Lehtio, J.
    et al.
    Wernérus, Henrik
    KTH, Superseded Departments, Biotechnology.
    Samuelson, Patrik
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Directed immobilization of recombinant staphylococci on cotton fibers by functional display of a fungal cellulose-binding domain2001In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 195, no 2, 197-204 p.Article in journal (Refereed)
    Abstract [en]

    The immobilization of recombinant staphylococci onto cellulose fibers through surface display of a fungal cellulose-binding domain (CBD) was investigated. Chimeric proteins containing the CBD from Trichoderma reesei cellulase Cel6A were found to be correctly targeted to the cell wall of Staphylococcus carnosus cells. since full-length proteins could be extracted and affinity-purified. Furthermore. surface accessibility of the CBD was verified using a monoclonal antibody and functionality in terms of cellulose-binding was demonstrated in two different assays in which recombinant staphylococci were found to efficiently bind to cotton fibers. The implications of this strategy of directed immobilization Tor the generation of whole-cell microbial tools Fur different applications will be discussed.

  • 49.
    Lönnberg, Hanna
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Fogelström, Linda
    Malmström, Eva
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    zhou, Q.
    Brumer, Harry
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Teeri, Tuula
    KTH, School of Biotechnology (BIO).
    Said, S.
    Berglund, L.
    Hult, Anders
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Grafting of poly(E-caprolactone) from microfibrillated cellulose films - for biocomposite applications2007In: Polymer Preprints, ISSN 0032-3934, Vol. 48, no 2, 409-410 p.Article in journal (Refereed)
  • 50.
    Lönnberg, Hanna
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Fogelström, Linda
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Malmström, Eva
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Samir, Said
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Berglund, Lars
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Hult, Anders
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    POLY 661-Grafting of poly(e-caprolactone) from microfibrillated cellulose films: for biocomposite applications2007Conference paper (Refereed)
12 1 - 50 of 90
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