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- Johansson, P., et al.
(författare)
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Crystallization and preliminary X-ray analysis of a xyloglucan endotransglycosylase from Populus tremula x tremuloides
- 2003
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 59, s. 535-537
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Tidskriftsartikel (refereegranskat)abstract
- 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).
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- Aspeborg, Henrik, et al.
(författare)
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Carbohydrate-active enzymes involved in the secondary cell wall biogenesis in hybrid aspen
- 2005
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 137:3, s. 983-997
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Baumann, Martin J., et al.
(författare)
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Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases : Biological implications for cell wall metabolism
- 2007
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Ingår i: The Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X. ; 19:6, s. 1947-1963
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Bollok, Monika, et al.
(författare)
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Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast Pichia pastoris
- 2005
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Ingår i: Applied Biochemistry and Biotechnology. - : Springer Science and Business Media LLC. - 0273-2289 .- 1559-0291. ; 126, s. 61-77
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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- Daniel, Geoffrey, et al.
(författare)
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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 microscopy
- 2006
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Ingår i: Holzforschung. - 0018-3830 .- 1437-434X. ; 60:6, s. 618-624
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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