Vincent G H Eijsink

Norwegian University of Life Sciences (NMBU), Aas, Akershus, Norway

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Publications (242)834.69 Total impact

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    ABSTRACT: Background Enzymes that degrade or modify polysaccharides are widespread in pro- and eukaryotes and have multiple biological roles and biotechnological applications. Recent advances in genome and secretome sequencing, together with associated bioinformatic tools, have enabled large numbers of carbohydrate-acting enzymes to be putatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of these enzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes. Results We have developed a new generation of multi-coloured chromogenic polysaccharide and protein substrates that can be used in cheap, convenient and high-throughput multiplexed assays. In addition, we have produced substrates of biomass materials in which the complexity of plant cell walls is partially maintained. Conclusions We show that these substrates can be used to screen the activities of glycosyl hydrolases, lytic polysaccharide monooxygenases and proteases and provide insight into substrate availability within biomass. We envisage that the assays we have developed will be used primarily for first-level screening of large numbers of putative carbohydrate-acting enzymes, and the assays have the potential to be incorporated into fully or semi-automated robotic enzyme screening systems. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0250-y) contains supplementary material, which is available to authorized users.
    Biotechnology for Biofuels 12/2015; 8(1). DOI:10.1186/s13068-015-0250-y · 6.04 Impact Factor
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    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. Current commercial enzyme cocktails contain LPMOs, which, considering the unique properties of these enzymes, may change optimal processing conditions. Here, we show that such modern cellulase cocktails release up to 60 % more glucose from a pretreated lignocellulosic substrate under aerobic conditions compared to anaerobic conditions. This higher yield correlates with the accumulation of oxidized products, which is a signature of LPMO activity. Spiking traditional cellulase cocktails with LPMOs led to increased saccharification yields, but only under aerobic conditions. LPMO activity on pure cellulose depended on the addition of an external electron donor, whereas this was not required for LPMO activity on lignocellulose. In this study, we demonstrate a direct correlation between saccharification yield and LPMO activity of commercial enzyme cocktails. Importantly, we show that the LPMO contribution to overall efficiency may be large if process conditions are adapted to the key determinants of LPMO activity, namely the presence of electron donors and molecular oxygen. Thus, the advent of LPMOs has a great potential, but requires rethinking of industrial bioprocessing procedures.
    Biotechnology for Biofuels 12/2015; 8(1). DOI:10.1186/s13068-015-0376-y · 6.04 Impact Factor
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    ABSTRACT: Bacteria and fungi express lytic polysaccharide monooxgyenase (LPMO) enzymes that act in conjunction with canonical hydrolytic sugar-processing enzymes to rapidly convert polysaccharides such as chitin, cellulose and starch to single monosaccharide products. In order to gain a better understanding of the structure and oxidative mechanism of these enzymes, large crystals (1–3 mm 3 ) of a chitin-processing LPMO from the Gram-positive soil bacterium Jonesia denitrificans were grown and screened for their ability to diffract neutrons. In addition to the collection of neutron diffraction data, which were processed to 2.1 Å resolution, a high-resolution room-temperature X-ray diffraction data set was collected and processed to 1.1 Å resolution in space group P 2 1 2 1 2 1 . To our knowledge, this work marks the first successful neutron crystallographic experiment on an LPMO. Joint X-ray/neutron refinement of the resulting data will reveal new details of the structure and mechanism of this recently discovered class of enzymes.
    Acta Crystallographica Section F: Structural Biology Communications 11/2015; 71(11):1448-1452. DOI:10.1107/S2053230X15019743
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    Katarzyna Kuczkowska · Geir Mathiesen · Vincent G H Eijsink · Inger Øynebråten ·
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    ABSTRACT: Background: Chemokines are attractive candidates for vaccine adjuvants due to their ability to recruit the immune cells. Lactic acid bacteria (LAB)-based delivery vehicles have potential to be used as a cheap and safe option for vaccination. Chemokine produced on the surface of LAB may potentially enhance the immune response to an antigen and this approach can be considered in development of future mucosal vaccines. Results: We have constructed strains of Lactobacillus plantarum displaying a chemokine on their surface. L. plantarum was genetically engineered to express and anchor to the surface a protein called CCL3Gag. CCL3Gag is a fusion protein comprising of truncated HIV-1 Gag antigen and the murine chemokine CCL3, also known as MIP-1α. Various surface anchoring strategies were explored: (1) a lipobox-based covalent membrane anchor, (2) sortase-mediated covalent cell wall anchoring, (3) LysM-based non-covalent cell wall anchoring, and (4) an N-terminal signal peptide-based transmembrane anchor. Protein production and correct localization were confirmed using Western blotting, flow cytometry and immunofluorescence microscopy. Using a chemotaxis assay, we demonstrated that CCL3Gag-producing L. plantarum strains are able to recruit immune cells in vitro. Conclusions: The results show the ability of engineered L. plantarum to produce a functional chemotactic protein immobilized on the bacterial surface. We observed that the activity of surface-displayed CCL3Gag differed depending on the type of anchor used. The chemokine which is a part of the bacteria-based vaccine may increase the recruitment of immune cells and, thereby, enhance the reaction of the immune system to the vaccine.
    Microbial Cell Factories 10/2015; 14(1):169. DOI:10.1186/s12934-015-0360-z · 4.22 Impact Factor
  • Oskar Bengtsson · Magnus Ø Arntzen · Geir Mathiesen · Morten Skaugen · Vincent G.H. Eijsink ·
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    ABSTRACT: Biological significance: The method described here enables rapid large-scale comparative studies of the secretomes of filamentous fungi and other microorganisms growing on a variety of solid substrates. This will in turn have implications for our understanding of enzymatic lignocellulose degradation, an area of significant scientific and industrial interest.
    Journal of proteomics 10/2015; DOI:10.1016/j.jprot.2015.10.017 · 3.89 Impact Factor
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    ABSTRACT: Given their safe use in humans and inherent adjuvanticity, Lactic Acid Bacteria may offer several advantages over other mucosal delivery strategies for cancer vaccines. The objective of this study is to evaluate the immune responses in mice after oral immunization with Lactobacillus (L) plantarum WCFS1 expressing a cell-wall anchored tumour antigen NY-ESO-1. And to investigate the immunostimulatory potency of this new candidate vaccine on human dendritic cells (DCs). L. plantarum displaying NY-ESO-1 induced NY-ESO-1 specific antibodies and T-cell responses in mice. By contrast, L. plantarum displaying either heat shock protein-27 or galectin-1, two conserved proteins, did not induce immunity, suggesting that immune tolerance to self-proteins cannot be broken by oral administration of L. plantarum. With respect to immunomodulation, immature DCs incubated with wild type or L. plantarum-NY-ESO-1 upregulated the expression of co-stimulatory molecules and secreted a large amount of interleukin (IL)-12, TNF-α, but not IL-4. Moreover, they upregulated the expression of immunosuppressive factors such as IL-10 and indoleamine 2,3-dioxygenase. Although L. plantarum-matured DCs expressed inhibitory molecules, they stimulated allogeneic T cells in-vitro. Collectively, the data indicate that L. plantarum-NY-ESO-1 can evoke antigen-specific immunity upon oral administration and induce DC maturation, raising the potential of its use in cancer immunotherapies.
    Human Vaccines & Immunotherapeutics 07/2015; DOI:10.1080/21645515.2015.1056952 · 2.37 Impact Factor
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    ABSTRACT: The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major im-portance for efficient processing of biomass. NcLP-MO9C from Neurospora crassa acts both on cellu-lose and on non-cellulose β-glucans, including cello-dextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extend-ed 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 interac-tions. Electron spin resonance (EPR) studies demon-strated that the Cu2+ center environment is altered upon substrate binding, whereas isothermal titration calorimetry (ITC) studies revealed binding affinities in the low micromolar range for polymeric substrates that are in part due to the presence of a carbohydrate-binding module (a CBM1). Importantly, the novel structure of NcLPMO9C enabled 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 sol-vent-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 C-1 and C4-oxidized products show an intermediate situation. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 07/2015; 290(38). DOI:10.1074/jbc.M115.660183 · 4.57 Impact Factor
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    Li Sun · Phillip B Pope · Vincent G H Eijsink · Anna Schnürer ·
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    ABSTRACT: Responses of bacterial and archaeal communities to the addition of straw during anaerobic digestion of manure at different temperatures (37°C, 44°C and 52°C) were investigated using five laboratory-scale semi-continuous stirred tank reactors. The results revealed that including straw as co-substrate decreased the species richness for bacteria, whereas increasing the operating temperature decreased the species richness for both archaea and bacteria, and also the evenness of the bacteria. Taxonomic classifications of the archaeal community showed that Methanobrevibacter dominated in the manure samples, while Methanosarcina dominated in all digesters regardless of substrate. Increase of the operating temperature to 52°C led to increased relative abundance of Methanoculleus and Methanobacterium. Among the bacteria, the phyla Firmicutes and Bacteroidetes dominated within all samples. Compared with manure itself, digestion of manure resulted in a higher abundance of an uncultured class WWE1 and lower abundance of Bacilli. Adding straw to the digesters increased the level of Bacteroidia, while increasing the operating temperature decreased the level of this class and instead increased the relative abundance of an uncultured genus affiliated to order MBA08 (Clostridia). A considerable fraction of bacterial sequences could not be allocated to genus level, indicating that novel phylotypes are resident in these communities. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.
    Microbial Biotechnology 07/2015; 8(5). DOI:10.1111/1751-7915.12298 · 3.21 Impact Factor
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    A E Naas · A K Mackenzie · B Dalhus · V G H Eijsink · P B Pope ·
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    ABSTRACT: Previous gene-centric analysis of a cow rumen metagenome revealed the first potentially cellulolytic polysaccharide utilization locus, of which the main catalytic enzyme (AC2aCel5A) was identified as a glycoside hydrolase (GH) family 5 endo-cellulase. Here we present the 1.8 Å three-dimensional structure of AC2aCel5A, and characterization of its enzymatic activities. The enzyme possesses the archetypical (β/α) 8-barrel found throughout the GH5 family, and contains the two strictly conserved catalytic glutamates located at the C-terminal ends of β-strands 4 and 7. The enzyme is active on insoluble cellulose and acts exclusively on linear β-(1,4)-linked glucans. Co-crystallization of a catalytically inactive mutant with substrate yielded a 2.4 Å structure showing cellotriose bound in the −3 to −1 subsites. Additional electron density was observed between Trp178 and Trp254, two residues that form a hydrophobic " clamp " , potentially interacting with sugars at the +1 and +2 subsites. The enzyme's active-site cleft was narrower compared to the closest structural relatives, which in contrast to AC2aCel5A, are also active on xylans, mannans and/or xyloglucans. Interestingly, the structure and function of this enzyme seem adapted to less-substituted substrates such as cellulose, presumably due to the insufficient space to accommodate the side-chains of branched glucans in the active-site cleft.
    Scientific Reports 07/2015; 5:11666. DOI:10.1038/srep11666 · 5.58 Impact Factor
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    ABSTRACT: Lactic acid bacteria (LAB) are promising vehicles for delivery of a variety of medicinal compounds, including antigens and cytokines. It has also been established that LAB are able to deliver cDNA to host cells. To increase the efficiency of LAB-driven DNA delivery we have constructed Lactobacillus plantarum strains targeting DEC-205, which is a receptor located at the surface of dendritic cells (DCs). The purpose was to increase uptake of bacterial cells, which could lead to improved cDNA delivery to immune cells. Anti-DEC-205 antibody (aDec) was displayed at the surface of L. plantarum using three different anchoring strategies: (1) covalent anchoring of aDec to the cell membrane (Lipobox domain, Lip); (2) covalent anchoring to the cell wall (LPXTG domain, CWA); (3) non-covalent anchoring to the cell wall (LysM domain, LysM). aDec was successfully expressed in all three strains, but surface location of the antibody could only be demonstrated for the two strains with cell wall anchors (CWA and LysM). Co-incubation of the engineered strains and DCs showed increased uptake when anchoring aDec using the CWA or LysM anchors. In a competition assay, free anti-DEC abolished the increased uptake, showing that the internalization is due to specific interactions between the DEC-205 receptor and aDec. To test plasmid transfer, a plasmid for expression of GFP under control of an eukaryotic promoter was transformed into the aDec expressing strains and GFP expression in DCs was indeed increased when using the strains producing cell-wall anchored aDec. Plasmid transfer to DCs in the gastro intestinal tract was also detected using a mouse model. Surprisingly, in mice the highest expression of GFP was observed for the strain in which aDec was coupled to the cell membrane. The results show that surface expression of aDec leads to increased internalization of L. plantarum and plasmid transfer in DCs and that efficiency depends on the type of anchor used. Interestingly, in vitro data indicates that cell wall anchoring is more effective, whereas in vivo data seem to indicate that anchoring to the cell membrane is preferable. It is likely that the more embedded localization of aDec in the latter case is favorable when cells are exposed to the harsh conditions of the gastro-intestinal tract.
    Microbial Cell Factories 07/2015; 14(1):95. DOI:10.1186/s12934-015-0290-9 · 4.22 Impact Factor
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    ABSTRACT: Human chitotriosidase (HCHT) is one of two active glycoside hydrolase family 18 chitinases produced by humans. The enzyme is associated with several diseases and is thought to play a role in the anti-parasite responses of the innate immune system. HCHT occurs in two isoforms, one 50 kDa (HCHT50) and one 39 kDa variant (HCHT39). Common for both isoforms is a catalytic domain with the (β/α)8 TIM barrel fold. HCHT50 has an additional linker-region, followed by a C-terminal carbohydrate-binding module (CBM) classified as CBM family 14 in the CAZy database. To gain further insight into enzyme functionality and especially the effect of the CBM, we expressed both isoforms and compared their catalytic properties on chitin and high molecular weight chitosans. HCHT50 degrades chitin faster than HCHT39 and much more efficiently. Interestingly, both HCHT50 and HCHT39 show biphasic kinetics on chitosan degradation where HCHT50 is faster initially and HCHT39 is faster in the second phase. Moreover, HCHT50 produces distinctly different oligomer distributions than HCHT39. This is likely due to increased transglycosylation activity for HCHT50 due the CBM extending the positive subsites binding surface and therefore promoting transglycosylation. Finally, studies with both chitin and chitosan showed that both isoforms have a similarly low degree of processivity. Combining functional and structural features of the two isoforms, it seems that HCHT combines features of exo-processive and endo-nonprocessive chitinases with the somewhat unusual CBM14 to reach a high degree of efficiency, in line with its alleged physiological task of being a "complete" chitinolytic machinery by itself. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 06/2015; 1854(10 Pt A). DOI:10.1016/j.bbapap.2015.06.008 · 4.66 Impact Factor
  • Anne Grethe Hamre · Daniel Schaupp · Vincent G H Eijsink · Morten Sørlie ·
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    ABSTRACT: The enzymatic degradation of the closely related insoluble polysaccharides; cellulose (β (1-4)-linked glucose) by cellulases and chitin (β (1-4)-linked N-acetylglucosamine) by chitinases, is of large biological and economical importance. Processive enzymes with different inherent directionalities, i.e. attacking the polysaccharide chains from opposite ends, are crucial for the efficiency of this degradation process. While processive cellulases with complementary functions differ in structure and catalytic mechanism, processive chitinases belong to one single protein family with similar active site architectures. Using the unique model system of Serratia marcescens with two processive chitinases attacking opposite ends of the substrate, we here show that different directionalities of processivity are correlated to distinct differences in the kinetic signatures for hydrolysis of oligomeric tetra-N-acetyl chitotetraose. Copyright © 2015. Published by Elsevier B.V.
    FEBS letters 05/2015; 589(15). DOI:10.1016/j.febslet.2015.05.034 · 3.17 Impact Factor
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    ABSTRACT: A new biogas process is initiated by adding a microbial community, typically in the form of a sample collected from a functional biogas plant. This inoculum has considerable impact on the initial performance of a biogas reactor, affecting parameters such as stability, biogas production yields and the overall efficiency of the anaerobic digestion process. In this study, we have analyzed changes in the microbial composition and performance of an inoculum during storage using barcoded pyrosequencing of bacterial and archaeal 16S ribosomal RNA (rRNA) genes, and determination of the biomethane potential, respectively. The inoculum was stored at room temperature, 4 and -20 °C for up to 11 months and cellulose was used as a standard substrate to test the biomethane potential. Storage up to 1 month resulted in similar final methane yields, but the rate of methane production was reduced by storage at -20 °C. Longer storage times resulted in reduced methane yields and slower production kinetics for all storage conditions, with room temperature and frozen samples consistently giving the best and worst performance, respectively. Both storage time and temperature affected the microbial community composition and methanogenic activity. In particular, fluctuations in the relative abundance of Bacteroidetes were observed. Interestingly, a shift from hydrogenotrophic methanogens to methanogens with the capacity to perform acetoclastic methanogensis was observed upon prolonged storage. In conclusion, this study suggests that biogas inocula may be stored up to 1 month with low loss of methanogenic activity, and identifies bacterial and archaeal species that are affected by the storage.
    Applied Microbiology and Biotechnology 05/2015; 99(13). DOI:10.1007/s00253-015-6623-0 · 3.34 Impact Factor
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    ABSTRACT: Enterococcus faecalis is a robust bacterium, which is able to survive in and adapt to hostile environments such as the urinary tract and bladder. In this label-free quantitative proteomic study based on MaxQuant LFQ algorithms, we identified 127 proteins present in the secretome of the clinical vancomycin-resistant isolate E. faecalis V583 and we compared proteins secreted in the initial phase of cultivation in urine with the secretome during cultivation in standard laboratory medium, 2xYT. Of the 54 identified proteins predicted to be secreted, six were exclusively found after cultivation in urine including the virulence factor EfaA ("endocarditis specific antigen") and its homologue EF0577 ("adhesion lipoprotein"). These two proteins are both involved in manganese transport, known to be an important determinant of colonization and infection, and may additionally function as adhesins. Other detected urine-specific proteins are involved in peptide transport (EF0063 and EF3106) and protease inhibition (EF3054). In addition, we found an uncharacterized protein (EF0764), which had not previously been linked to the adaptation of V583 to a urine environment, and which is unique to E. faecalis. Proteins found in both environments included a histone-like protein, EF1550, that was up-regulated during cultivation in urine and that has a homologue in streptococci (HlpA) known to be involved in bacterial adhesion to host cells. Up-regulated secreted proteins included autolysins. These results from secretome analyses are largely compatible with previously published data from transcriptomics studies. All in all, the present data indicate that transport, in particular metal transport, adhesion, cell wall remodelling and the unknown function carried out by the unique EF0764 are important for enterococcal adaptation to the urine environment. These results provide a basis for a more targeted exploration of novel proteins involved in the adaptability and pathogenicity of E. faecalis.
    PLoS ONE 04/2015; 10(4):e0126694. DOI:10.1371/journal.pone.0126694 · 3.23 Impact Factor

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    ABSTRACT: Group I grass pollen allergens are major contributors to grass pollen-related seasonal allergic rhinitis, and as such a primary target for allergen specific immunotherapy. In this study the potential therapeutic role of oral application of Lactobacillus plantarum WCFS1, directing cell wall attachment of the recombinant Fes p 1 allergen, from Festuca pratensis was tested in a mouse model of Fes p 1 allergy. For surface expression of Fes p 1 allergen in L. plantarum WCFS1 pSIP system with inducible expression was used. Balb/c mice were sensitized with Fes p 1 protein in alum and subsequently received live recombinant L. plantarum orally. Antibody levels (IgE, total IgG, IgG1, IgG2a, and IgA) were determined by ELISA. Differential eosinophil count in peripheral blood was performed. Reduced peripheral blood eosinophilia and increased serum IgG2A levels was detected in both groups which received live L. plantarum orally. Specific serum IgA levels were increased only in mice treated with the recombinant bacteria. Oral application of L. plantarum WCFS1 has a beneficial therapeutic effect in a mouse model of Fes p 1 allergy. Cell surface expression of Fes p 1 allergen potentiates this phenomenon in an allergen specific way. Copyright © 2015. Published by Elsevier B.V.
    Journal of Biotechnology 02/2015; 199. DOI:10.1016/j.jbiotec.2015.01.028 · 2.87 Impact Factor
  • Ingrid Lea Karlskås · Zhian Saleihan · Helge Holo · Geir Mathiesen · Vincent G H Eijsink ·
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    ABSTRACT: One of the ABC transporter systems in E. faecalis V583 is encoded by the ef0176-ef0180 gene cluster, which differs from orthologous operons in related bacteria in that it contains two genes putatively encoding substrate-binding proteins (SBPs). These SBPs, EF0176 and EF0177, have previously been identified on the surface of E. faecalis. By phenotypic studies of single and double knock-out mutants we show here that EF0176 and EF0177 are specific for ribonucleosides and, by inference, that the EF0176-EF0180 ABC transporter plays a role in nucleoside uptake. The specificity of the SBPs was mapped using growth experiments on a medium, RPMI-1640, that only supports growth of E. faecalis when supplemented with purine nucleosides or their corresponding bases. This analysis was complemented by studies with toxic fluorinated pyrimidine ribonucleoside analogues and competition experiments. The data show that EF0176 and EF0177 have broad, overlapping, but not identical substrate specificities and that they, together, are likely to bind and facilitate the transport of all common ribonucleosides. Comparative sequence analysis and inspection of an available crystal structure of an orthologue, PnrA from Treponema pallidum, showed that the strongest binding interactions between the protein and the ligand involve the ribose moiety and that sequence variation in the binding site primarily affects interactions with the base. This explains both the broad substrate specificity of these binding proteins and the observed variations therein. The presence of two SBPs in this nucleoside ABC transporter system in E. faecalis may improve the bacterium's ability to scavenge nucleosides.
    Microbiology 01/2015; 161(Pt_4). DOI:10.1099/mic.0.000045 · 2.56 Impact Factor
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    ABSTRACT: The lytic polysaccharide monooxygenases (LPMOs) have received considerable attention after their discovery in 2010 due to their ability to boost the enzymatic conversion of recalcitrant polysaccharides. Here, we describe the enzymatic properties of SgLPMO10F, a small (15 kDa) auxilliary activity family 10 (AA10) LPMO from Streptomyces griseus belonging to a clade of the phylogenetic tree without any characterized representative. The protein was expressed using a Brevibacillus-based expression system that had not been used previously for LPMO expression and that ensures correct processing of the N-terminus that is crucial for LPMO activity. The enzyme was active towards both α- and β-chitin and showed stronger binding and more release of soluble oxidized products for the latter allomorph. In chitinase synergy assays, however, SgLPMO10F worked slightly better for α-chitin, increasing chitin solubilization yields up to ~30-fold and ~20-fold for α- and β-chitin, respectively. Synergy experiments with various chitinases showed that addition of SgLPMO10F leads to a substantial increase in the (GlcNAc)2 :GlcNAc product ratio, in reactions with α-chitin only. This underpins the structural differences between the substrates and also shows that, on α-chitin, SgLPMO10F affects the binding mode and/or degree of processivity of the chitinases tested. Variation in the only exposed aromatic residue in the substrate-binding surface of LPMO10s has previously been linked to preferential binding for α-chitin (exposed Trp) or β-chitin (exposed Tyr). Mutation of this residue, Tyr56, in SgLPMO10F to Trp had no detectable effect on substrate binding preferences, but in synergy experiments the mutant seemed more efficient on α-chitin. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    FEBS Journal 01/2015; 282(6). DOI:10.1111/febs.13203 · 4.00 Impact Factor
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    Vivekanand Vivekanand · Elisabeth F. Olsen · Vincent G.H. Eijsink · Svein Jarle Horn ·

    Bioresources 11/2014; 9(1). DOI:10.15376/biores.9.1.1311-1324 · 1.43 Impact Factor
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    ABSTRACT: Recent metagenomic analyses have identified uncultured bacteria that are abundant in the rumen of herbivores and which possess putative biomass-converting enzyme systems. Here we investigate the saccharolytic capabilities of a Polysaccharide Utilization Locus (PUL) that has been reconstructed from an uncultured Bacteroidetes phylotype (SRM-1) that dominates the rumen microbiome of Arctic reindeer. Characterization of the three PUL-encoded outer membrane glycoside hydrolases was performed using chromogenic substrates for initial screening, followed by detailed analyses of products generated from selected substrates, using high-pressure anion-exchange chromatography with electrochemical detection. Two GH5 endoglucanases (GH5_g and GH5_h) demonstrated activity against β-glucans, xylans and xyloglucan, whereas GH5_h and the third enzyme, GH26_i, were active on several mannan substrates. Synergy experiments examining different combinations of the three enzymes demonstrated limited activity enhancement on individual substrates. Binding analysis of a SusE-positioned lipoprotein revealed an affinity towards β-glucans and, to a lesser extent mannan, but unlike the two SusD-like lipoproteins previously characterised from the same PUL, binding to cellulose was not observed. Overall, these activities and binding specificities correlated well with the glycan content of the reindeer rumen which was determined using comprehensive microarray polymer profiling and showed abundance of various hemicellulose glycans. The substrate versatility of this single PUL putatively expands our perceptions regarding PUL machineries, which so far have demonstrated a "one cognate PUL for each substrate" type of gene organization. The presence of a PUL that possesses saccharolytic activity against a mixture of abundantly available polysaccharides supports SRM-1 dominance in the Svalbard reindeer rumen microbiome.
    Applied and Environmental Microbiology 10/2014; 81(1). DOI:10.1128/AEM.02858-14 · 3.67 Impact Factor

Publication Stats

9k Citations
834.69 Total Impact Points


  • 2008-2015
    • Norwegian University of Life Sciences (NMBU)
      • Department of Chemistry, Biotechnology and Food Science (IKBM)
      Aas, Akershus, Norway
  • 2013
    • Swedish University of Agricultural Sciences
      Uppsala, Uppsala, Sweden
  • 2012
    • Drew University
      Madison, New Jersey, United States
    • Oslo University Hospital
      • Department of Medical Biochemistry
      Kristiania (historical), Oslo, Norway
  • 2010
    • Stord/Haugesund University College
      Haugesund, Rogaland, Norway
    • University of Aberdeen
      • Department of Chemistry
      Aberdeen, Scotland, United Kingdom
  • 2004-2010
    • Universität Potsdam
      • Institute of Chemistry
      Potsdam, Brandenburg, Germany
  • 1992-2010
    • University of Groningen
      • Department of Genetics
      Groningen, Groningen, Netherlands
    • European Molecular Biology Laboratory
      Heidelburg, Baden-Württemberg, Germany
  • 2006
    • Norwegian University of Science and Technology
      • Department of Physics
      Trondheim, Sor-Trondelag Fylke, Norway
  • 2002-2005
    • University of Dundee
      • Division of Molecular Microbiology
      Dundee, Scotland, United Kingdom
  • 1998-2002
    • University of Oslo
      • Department of Biochemistry
      Oslo, Oslo, Norway
  • 1997
    • Martin Luther University of Halle-Wittenberg
      • Institut für Biologie
      Halle, Saxony-Anhalt, Germany
  • 1995
    • University of Birmingham
      Birmingham, England, United Kingdom
  • 1993
    • IMSA Amsterdam
      Amsterdamo, North Holland, Netherlands