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| 2. |
- Frandsen, K. E. H., et al.
(författare)
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The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases
- 2016
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Ingår i: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 12:4, s. 298-
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Tidskriftsartikel (refereegranskat)abstract
- Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.
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| 3. |
- Gloster, Tracey M., et al.
(författare)
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Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12
- 2007
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 282:26, s. 19177-19189
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Tidskriftsartikel (refereegranskat)abstract
- The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed hemicellulose. One such hemicellulose is xyloglucan, which displays a beta-1,4-linked D-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand free and xyloglucan- oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (beta/alpha)(8) and beta-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the beta-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzymeis similarly competent in the degradation of unbranched glucans. In the case of the (beta/alpha)(8) GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.
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| 5. |
- Hernández-Rollán, Cristina, et al.
(författare)
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LyGo : A Platform for Rapid Screening of Lytic Polysaccharide Monooxygenase Production
- 2021
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 10:4, s. 897-906
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Tidskriftsartikel (refereegranskat)abstract
- Environmentally friendly sources of energy and chemicals are essential constituents of a sustainable society. An important step toward this goal is the utilization of biomass to supply building blocks for future biorefineries. Lytic polysaccharide monooxygenases (LPMOs) are enzymes that play a critical role in breaking the chemical bonds in the most abundant polymers found in recalcitrant biomass, such as cellulose and chitin. To use them in industrial processes they need to be produced in high titers in cell factories. Predicting optimal strategies for producing LPMOs is often nontrivial, and methods allowing for screening several strategies simultaneously are therefore needed. Here, we present a standardized platform for cloning LPMOs. The platform allows users to combine gene fragments with 14 different expression vectors in a simple 15 min reaction, thus enabling rapid exploration of several gene contexts, hosts, and expression strategies in parallel. The open-source LyGo platform is accompanied by easy-to-follow online protocols for both cloning and expression. As a demonstration of its utility, we explore different strategies for expressing several different LPMOs in Escherichia coli, Bacillus subtilis, and Komagataella phaffii.
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| 6. |
- Krogh, Kristian B. R. M., et al.
(författare)
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Characterization and kinetic analysis of a thermostable GH3 beta-glucosidase from Penicillium brasilianum
- 2010
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 86:1, s. 143-154
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Tidskriftsartikel (refereegranskat)abstract
- A GH3 beta-glucosidase (BGL) from Penicillium brasilianum was purified to homogeneity after cultivation on a cellulose and xylan rich medium. The BGL was identified in a genomic library, and it was successfully expressed in Aspergillus oryzae. The BGL had excellent stability at elevated temperatures with no loss in activity after 24 h of incubation at 60A degrees C at pH 4-6, and the BGL was shown to have significantly higher stability at these conditions in comparison to Novozym 188 and to other fungal GH3 BGLs reported in the literature. The BGL had significant lower affinity for cellobiose compared with the artificial substrate para-nitrophenyl-beta-d-glucopyranoside (pNP-Glc) and further, pronounced substrate inhibition using pNP-Glc. Kinetic studies demonstrated the high importance of using cellobiose as substrate and glucose as inhibitor to describe the inhibition kinetics of BGL taking place during cellulose hydrolysis. A novel assay was developed to characterize this glucose inhibition on cellobiose hydrolysis. The assay uses labelled glucose-C-13(6) as inhibitor and subsequent mass spectrometry analysis to quantify the hydrolysis rates.
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| 7. |
- Salomon Johansen, Katja, 1969, et al.
(författare)
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Harnessing the potential of LPMO-containing cellulase cocktails poses new demands on processing conditions
- 2015
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 8:1, s. 1-
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Tidskriftsartikel (refereegranskat)abstract
- The emerging bioeconomy depends on improved methods for processing of lignocellulosic biomass to fuels and chemicals. Saccharification of lignocellulose to fermentable sugars is a key step in this regard where enzymatic catalysis plays an important role and is a major cost driver. Traditionally, enzyme cocktails for the conversion of cellulose to fermentable sugars mainly consisted of hydrolytic cellulases. However, the recent discovery of lytic polysaccharide monooxygenases (LPMOs), which cleave cellulose using molecular oxygen and an electron donor, has provided new tools for biomass saccharification
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