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| 2. |
- Desmet, Tom, et al.
(författare)
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Synthesis and Evaluation of 2-Deoxy-2-amino-beta-cellobiosides as Cellulase Inhibitors
- 2010
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Ingår i: Journal of carbohydrate chemistry. - : Informa UK Limited. - 0732-8303 .- 1532-2327. ; 29:4, s. 164-180
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Tidskriftsartikel (refereegranskat)abstract
- The cellulase mixture of Hypocrea jecorina (formerly Trichoderma reesei) contains a variety of exo- and endoglucanases that belong to different structural families. As such, these enzymes form an interesting model system to study the enzyme-ligand interactions in glycoside hydrolases. The nucleophilic carboxylate of retaining -glycosidases is believed to form a hydrogen bond with the 2-hydroxyl group of their substrate. Consequently, replacing this hydroxyl group with an amino group should result in a stronger electrostatic interaction and thus an increased affinity for the ligand. In this study, several modified cellobiosides were synthesized and evaluated as cellulase inhibitors. The introduction of an amino group was found to have an unpredictable effect on the inhibitory power of the ligands. However, the enzymes display a very high affinity for the corresponding 2-azido compounds, precursors in the synthetic route. The new ligand m-iodobenzyl 2-deoxy-2-azido--cellobioside even is the strongest inhibitor of cellobiohydrolase I known to date (KI= 1 M).
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| 3. |
- Jonsson Rudsander, Ulla, et al.
(författare)
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Comparative NMR analysis of cellooligosaccharide hydrolysis by GH9 bacterial and plant endo-1,4-ss-glucanases
- 2008
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 47:18, s. 5235-5241
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Kallas, Åsa M., et al.
(författare)
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Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula x tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris
- 2005
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Ingår i: Biochemical Journal. - 0264-6021. ; 390, s. 105-113
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Koivula, Anu, et al.
(författare)
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The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175.
- 2002
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Ingår i: J Am Chem Soc. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 124:34, s. 10015-24
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Nishikubo, Nobuyuki, et al.
(författare)
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Xyloglucan endo-Transglycosylase-Mediated Xyloglucan Rearrangements in Developing Wood of Hybrid Aspen
- 2011
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 155:1, s. 399-413
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Tidskriftsartikel (refereegranskat)abstract
- Xyloglucan endo-transglycosylases (XETs) encoded by xyloglucan endo-transglycosylases/hydrolase (XTH) genes modify the xyloglucan-cellulose framework of plant cell walls, thereby regulating their expansion and strength. To evaluate the importance of XET in wood development, we studied xyloglucan dynamics and XTH gene expression in developing wood and modified XET activity in hybrid aspen (Populus tremula X tremuloides) by overexpressing PtxtXET16-34. We show that developmental modifications during xylem differentiation include changes from loosely to tightly bound forms of xyloglucan and increases in the abundance of fucosylated xyloglucan epitope recognized by the CCRC-M1 antibody. We found that at least 16 Populus XTH genes, all likely encoding XETs, are expressed in developing wood. Five genes were highly and ubiquitously expressed, whereas PtxtXET16-34 was expressed more weakly but specifically in developing wood. Transgenic up-regulation of XET activity induced changes in cell wall xyloglucan, but its effects were dependent on developmental stage. For instance, XET overexpression increased abundance of the CCRC-M1 epitope in cambial cells and xylem cells in early stages of differentiation but not in mature xylem. Correspondingly, an increase in tightly bound xyloglucan content was observed in primary-walled xylem but a decrease was seen in secondary-walled xylem. Thus, in young xylem cells, XET activity limits xyloglucan incorporation into the tightly bound wall network but removes it from cell walls in older cells. XET overexpression promoted vessel element growth but not fiber expansion. We suggest that the amount of nascent xyloglucan relative to XET is an important determinant of whether XET strengthens or loosens the cell wall.
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| 7. |
- Passoth, Volkmar, et al.
(författare)
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Airtight storage of moist wheat grain improves bioethanol yields
- 2009
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Ingår i: Biotechnology for Biofuels. - London, United Kingdom : Springer Science and Business Media LLC. - 1754-6834. ; 2
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Tidskriftsartikel (refereegranskat)abstract
- Background: Drying is currently the most frequently used conservation method for cereal grain, which in temperate climates consumes a major part of process energy. Airtight storage of moist feed grain using the biocontrol yeast Pichia anomala as biopreservation agent can substantially reduce the process energy for grain storage. In this study we tested the potential of moist stored grain for bioethanol production.Results: The ethanol yield from moist wheat was enhanced by 14% compared with the control obtained from traditionally (dry) stored grain. This enhancement was observed independently of whether or not P. anomala was added to the storage system, indicating that P. anomala does not impair ethanol fermentation. Starch and sugar analyses showed that during pre-treatment the starch of moist grain was better degraded by amylase treatment than that of the dry grain. Additional pre-treatment with cellulose and hemicellulose-degrading enzymes did not further increase the total ethanol yield. Sugar analysis after this pre-treatment showed an increased release of sugars not fermentable by Saccharomyces cerevisiae.Conclusion: The ethanol yield from wheat grain is increased by airtight storage of moist grain, which in addition can save substantial amounts of energy used for drying the grain. This provides a new opportunity to increase the sustainability of bioethanol production.
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| 8. |
- Piens, Kathleen, et al.
(författare)
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Glycosynthase activity of hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 nucleophile mutants
- 2007
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Ingår i: Organic and Biomolecular Chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 5:24
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Tidskriftsartikel (refereegranskat)abstract
- Glycosynthases are active-site mutants of glycoside hydrolases that catalyse glycosyl transfer using suitable activated donor substrates without competing product hydrolysis ( S. M. Hancock, M. D. Vaughan and S. G. Withers, Curr. Opin. Chem. Biol., 2006, 10, 509-519). Site-directed mutagenesis of the catalytic nucleophile, Glu-85, of a Populus tremula x tremuloides xyloglucan endo-transglycosylase (PttXET16-34, EC 2.4.1.207) into alanine, glycine, and serine yielded enzymes with glycosynthase activity. Product analysis indicated that PttXET16-34 E85A in particular was able to catalyse regio- and stereospecific homo- and hetero- condensations of alpha-xylogluco-oligosaccharyl fluoride donors XXXG alpha F andXLLG alpha F to produce xyloglucans with regular sidechain substitution patterns. This substrate promiscuity contrasts that of the Humicola insolens Ce17B E197A glycosynthase, which was not able to polymerise the di-galactosylated substrate XLLG alpha F. The production of the PttXET16-34 E85A xyloglucosynthase thus expands the repertoire of glycosynthases to include those capable of synthesising structurally homogenenous xyloglucans
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| 9. |
- Piens, Kathleen, et al.
(författare)
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Mechanism-based labeling defines the free energy change for formation of the covalent glycosyl-enzyme intermediate in a xyloglucan endo-transglycosylase
- 2008
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 283:32, s. 21864-21872
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Tidskriftsartikel (refereegranskat)abstract
- Xyloglucan endo-transglycosylases (XETs) are key enzymes involved in the restructuring of plant cell walls during morphogenesis. As members of glycoside hydrolase family 16 (GH16), XETs are predicted to employ the canonical retaining mechanism of glycosyl transfer involving a covalent glycosyl-enzyme intermediate. Here, we report the accumulation and direct observation of such intermediates of PttXET16-34 from hybrid aspen by electrospray mass spectrometry in combination with synthetic "blocked" substrates, which function as glycosyl donors but are incapable of acting as glycosyl acceptors. Thus, GalGXXXGGG and GalGXXXGXXXG react with the wild-type enzyme to yield relatively stable, kinetically competent, covalent GalG-enzyme and GalGXXXG-enzyme complexes, respectively (Gal = Gal beta(1 -> 4), G = Glc beta(1 -> 4), and X = Xyl alpha(1 -> 6) Glc beta(1 -> 4)). Quantitation of ratios of protein and saccharide species at pseudo-equilibrium allowed us to estimate the free energy change (Delta G(0)) for the formation of the covalent GalGXXXG-enzyme as 6.3-8.5 kJ/mol (1.5-2.0 kcal/mol). The data indicate that the free energy of the beta(1 -> 4) glucosidic bond in xyloglucans is preserved in the glycosyl-enzyme intermediate and harnessed for religation of the polysaccharide in vivo.
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