| 1. |
- Borisova, Anna, et al.
(författare)
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The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolyses of p-nitrophenyl glycosides.
- 2015
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Ingår i: Carbohydrate Research. - : Elsevier BV. - 1873-426X .- 0008-6215. ; 412, s. 43-49
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Tidskriftsartikel (refereegranskat)abstract
- In the present work we suggest an efficient method, using the whole time course of the reaction, whereby parameters kcat, Km and product KI for the hydrolysis of a p-nitrophenyl glycoside by an exo-acting glycoside hydrolase can be estimated in a single experiment. Its applicability was demonstrated for three retaining exo-glycoside hydrolases, β-xylosidase from Aspergillus awamori, β-galactosidase from Penicillium sp. and α-galactosidase from Thermotoga maritima (TmGalA). During the analysis of the reaction course catalyzed by the TmGalA enzyme we had observed that a non-enzymatic process, mutarotation of the liberated α-d-galactose, affected the reaction significantly.
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| 2. |
- Pearce, Elena A., et al.
(författare)
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Substantial light woodland and open vegetation characterized the temperate forest biome before Homo sapiens
- 2023
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Ingår i: Science Advances. - 2375-2548. ; 9:45
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Tidskriftsartikel (refereegranskat)abstract
- The extent of vegetation openness in past European landscapes is widely debated. In particular, the temperate forest biome has traditionally been defined as dense, closed-canopy forest; however, some argue that large herbivores maintained greater openness or even wood-pasture conditions. Here, we address this question for the Last Interglacial period (129,000–116,000 years ago), before Homo sapiens–linked megafauna declines and anthropogenic landscape transformation. We applied the vegetation reconstruction method REVEALS to 96 Last Interglacial pollen records. We found that light woodland and open vegetation represented, on average, more than 50% cover during this period. The degree of openness was highly variable and only partially linked to climatic factors, indicating the importance of natural disturbance regimes. Our results show that the temperate forest biome was historically heterogeneous rather than uniformly dense, which is consistent with the dependency of much of contemporary European biodiversity on open vegetation and light woodland.
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| 3. |
- Borisova, Anna, et al.
(författare)
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alpha-Galactobiosyl units: thermodynamics and kinetics of their formation by transglycosylations catalysed by the GH36 alpha-galactosidase from Thermotoga maritima
- 2015
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Ingår i: Carbohydrate Research. - : Elsevier BV. - 0008-6215 .- 1873-426X. ; 401, s. 115-121
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Tidskriftsartikel (refereegranskat)abstract
- Broad regioselectivity of a-galactosidase from Thermotoga maritima (pTmGal36A) is a limiting factor for application of the enzyme in the directed synthesis of oligogalactosides. However, this property can be used as a convenient tool in studies of thermodynamics of a glycosidic bond. Here, a novel approach to energy difference estimation is suggested. Both transglycosylation and hydrolysis of three types of galactosidic linkages were investigated using total kinetics of formation and hydrolysis of pNP-galacto-biosides catalysed by monomeric glycoside hydrolase family 36 a-galactosidase from T. maritima, a retaining exo-acting glycoside hydrolase. We have estimated transition state free energy differences between the 1,2-and 1,3-linkage (Delta Delta G(0)(double dagger) values were equal 5.34 +/- 0.85 kJ/ mol) and between 1,6-linkage and 1,3-linkage (Delta Delta G(0)(double dagger) = 1.46 +/- 0.23 kJ/mol) in pNP-galactobiosides over the course of the reaction catalysed by TmGal36A. Using the free energy difference for formation and hydrolysis of glycosidic linkages (Delta Delta G(F)(double dagger) -Delta Delta G(H)(double dagger)), we found that the 1,2-linkage was 2.93 +/- 0.47 kJ/ mol higher in free energy than the 1,3-linkage, and the 1,6-linkage 4.44 +/- 0.71 kJ/ mol lower. (C) 2014 Elsevier Ltd. All rights reserved.
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| 4. |
- Borisova, Anna, et al.
(författare)
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Correlation of structure, function and protein dynamics in GH7 cellobiohydrolases from Trichoderma atroviride, T. reesei and T. harzianum
- 2018
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Background: The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, similar to 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate.Results: The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. The catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs.Conclusions: We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.
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| 5. |
- Borisova, Anna, et al.
(författare)
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Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum 3C
- 2015
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Ingår i: FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 282, s. 4515-4537
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Tidskriftsartikel (refereegranskat)abstract
- The ascomycete Geotrichum candidum is a versatile and efficient decay fungus that is involved, for example, in biodeterioration of compact discs; notably, the 3C strain was previously shown to degrade filter paper and cotton more efficiently than several industrial enzyme preparations. Glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs) are the primary constituents of industrial cellulase cocktails employed in biomass conversion, and feature tunnel-enclosed active sites that enable processive hydrolytic cleavage of cellulose chains. Understanding the structure-function relationships defining the activity and stability of GH7 CBHs is thus of keen interest. Accordingly, we report the comprehensive characterization of the GH7 CBH secreted by G. candidum (GcaCel7A). The bimodular cellulase consists of a family 1 cellulose-binding module (CBM) and linker connected to a GH7 catalytic domain that shares 64% sequence identity with the archetypal industrial GH7 CBH of Hypocrea jecorina (HjeCel7A). GcaCel7A shows activity on Avicel cellulose similar to HjeCel7A, with less product inhibition, but has a lower temperature optimum (50 degrees C versus 60-65 degrees C, respectively). Five crystal structures, with and without bound thio-oligosaccharides, show conformational diversity of tunnel-enclosing loops, including a form with partial tunnel collapse at subsite -4 not reported previously in GH7. Also, the first O-glycosylation site in a GH7 crystal structure is reported - on a loop where the glycan probably influences loop contacts across the active site and interactions with the cellulose surface. The GcaCel7A structures indicate higher loop flexibility than HjeCel7A, in accordance with sequence modifications. However, GcaCel7A retains small fluctuations in molecular simulations, suggesting high processivity and low endo-initiation probability, similar to HjeCel7A.
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| 6. |
- Borisova, Anna, et al.
(författare)
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Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity
- 2015
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290, s. 22955-22969
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Tidskriftsartikel (refereegranskat)abstract
- The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose beta-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu2+-center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9Cenabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4- oxidized products show an intermediate situation.
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| 7. |
- Burkhard, Benjamin, et al.
(författare)
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Mapping and assessing ecosystem services in the EU - Lessons learned from the ESMERALDA approach of integration
- 2018
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Ingår i: One Ecosystem. - : Pensoft Publishers. - 2367-8194. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- The European Union (EU) Horizon 2020 Coordination and Support Action ESMERALDA aimed at developing guidance and a flexible methodology for Mapping and Assessment of Ecosystems and their Services (MAES) to support the EU member states in the implementation of the EU Biodiversity Strategy’s Target 2 Action 5. ESMERALDA’s key tasks included network creation, stakeholder engagement, enhancing ecosystem services mapping and assessment methods across various spatial scales and value domains, work in case studies and support of EU member states in MAES implementation. Thus ESMERALDA aimed at integrating various project outcomes around four major strands: i) Networking, ii) Policy, iii) Research and iv) Application. The objective was to provide guidance for integrated ecosystem service mapping and assessment that can be used for sustainable decision-making in policy, business, society, practice and science at EU, national and regional levels. This article presents the overall ESMERALDA approach of integrating the above-mentioned project components and outcomes and provides an overview of how the enhanced methods were applied and how they can be used to support MAES implementation in the EU member states. Experiences with implementing such a large pan-European Coordination and Support Action in the context of EU policy are discussed and recommendations for future actions are given.
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| 8. |
- Githumbi, Esther, et al.
(författare)
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European pollen-based REVEALS land-cover reconstructions for the Holocene : Methodology, mapping and potentials
- 2022
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Ingår i: Earth System Science Data. - : Copernicus GmbH. - 1866-3508 .- 1866-3516. ; 14:4, s. 1581-1619
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Tidskriftsartikel (refereegranskat)abstract
- Quantitative reconstructions of past land cover are necessary to determine the processes involved in climate-human-land-cover interactions. We present the first temporally continuous and most spatially extensive pollen-based land-cover reconstruction for Europe over the Holocene (last 11g€¯700g€¯calg€¯yrg€¯BP). We describe how vegetation cover has been quantified from pollen records at a 11 spatial scale using the "Regional Estimates of VEgetation Abundance from Large Sites"(REVEALS) model. REVEALS calculates estimates of past regional vegetation cover in proportions or percentages. REVEALS has been applied to 1128 pollen records across Europe and part of the eastern Mediterranean-Black Sea-Caspian corridor (30-75° N, 25° W-50° E) to reconstruct the percentage cover of 31 plant taxa assigned to 12 plant functional types (PFTs) and 3 land-cover types (LCTs). A new synthesis of relative pollen productivities (RPPs) for European plant taxa was performed for this reconstruction. It includes multiple RPP values (≥2 values) for 39 taxa and single values for 15 taxa (total of 54 taxa). To illustrate this, we present distribution maps for five taxa (Calluna vulgaris, Cerealia type (t)., Picea abies, deciduous Quercus t. and evergreen Quercus t.) and three land-cover types (open land, OL; evergreen trees, ETs; and summer-green trees, STs) for eight selected time windows. The reliability of the REVEALS reconstructions and issues related to the interpretation of the results in terms of landscape openness and human-induced vegetation change are discussed. This is followed by a review of the current use of this reconstruction and its future potential utility and development. REVEALS data quality are primarily determined by pollen count data (pollen count and sample, pollen identification, and chronology) and site type and number (lake or bog, large or small, one site vs. multiple sites) used for REVEALS analysis (for each grid cell). A large number of sites with high-quality pollen count data will produce more reliable land-cover estimates with lower standard errors compared to a low number of sites with lower-quality pollen count data. The REVEALS data presented here can be downloaded from https://doi.org/10.1594/PANGAEA.937075 (Fyfe et al., 2022).
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| 9. |
- Haddad Momeni, Majid, et al.
(författare)
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Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life
- 2016
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 82, s. 3395-3409
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Tidskriftsartikel (refereegranskat)abstract
- Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes commonly employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba: Dictyostelium discoideum Cel7A (DdiCel7A) and Dictyostelium purpureum Cel7A (DpuCel7A). DdiCel7A and DpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures of DdiCel7A and DpuCel7A, resolved to 2.1 angstrom and 2.7 angstrom respectively, are homologous to those of other GH7 CBHs with an enclosed active-site tunnel. Two primary differences between the Dictyostelium CBHs and the archetypal model GH7 CBH, Trichoderma reesei Cel7A (TreCel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity of TreCel7A, the family 1 TreCel7A CBM and linker were added to the C terminus of each of the Dictyostelium enzymes, creating DdiCel7A(CBM) and DpuCel7A(CBM), which were recombinantly expressed in T. reesei. DdiCel7A(CBM) and DpuCel7A(CBM) hydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently as TreCel7A when hydrolysis was compared at their temperature optima. The K-i of cellobiose was significantly higher for DdiCel7A(CBM) and DpuCel7A(CBM) than for TreCel7A: 205, 130, and 29 mu M, respectively. Taken together, the present study highlights the remarkable degree of conservation of the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life.IMPORTANCEGH7 CBHs are among the most important cellulolytic enzymes both in nature and for emerging industrial applications for cellulose breakdown. Understanding the diversity of these key industrial enzymes is critical to engineering them for higher levels of activity and greater stability. The present work demonstrates that two GH7 CBHs from social amoeba are surprisingly quite similar in structure and activity to the canonical GH7 CBH from the model biomass-degrading fungus T. reesei when tested under equivalent conditions (with added CBM-linker domains) on an industrially relevant substrate.
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| 10. |
- Serge, M. A., et al.
(författare)
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Testing the Effect of Relative Pollen Productivity on the REVEALS Model : A Validated Reconstruction of Europe-Wide Holocene Vegetation
- 2023
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Ingår i: Land. - : MDPI. - 2073-445X. ; 12:5
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Tidskriftsartikel (refereegranskat)abstract
- Reliable quantitative vegetation reconstructions for Europe during the Holocene are crucial to improving our understanding of landscape dynamics, making it possible to assess the past effects of environmental variables and land-use change on ecosystems and biodiversity, and mitigating their effects in the future. We present here the most spatially extensive and temporally continuous pollen-based reconstructions of plant cover in Europe (at a spatial resolution of 1 degrees x 1 degrees) over the Holocene (last 11.7 ka BP) using the 'Regional Estimates of VEgetation Abundance from Large Sites' (REVEALS) model. This study has three main aims. First, to present the most accurate and reliable generation of REVEALS reconstructions across Europe so far. This has been achieved by including a larger number of pollen records compared to former analyses, in particular from the Mediterranean area. Second, to discuss methodological issues in the quantification of past land cover by using alternative datasets of relative pollen productivities (RPPs), one of the key input parameters of REVEALS, to test model sensitivity. Finally, to validate our reconstructions with the global forest change dataset. The results suggest that the RPPs.st1 (31 taxa) dataset is best suited to producing regional vegetation cover estimates for Europe. These reconstructions offer a long-term perspective providing unique possibilities to explore spatial-temporal changes in past land cover and biodiversity.
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