Publications by authors named "Finn Lillelund Aachmann"

21 Publications

  • Page 1 of 1

Alginate hydrogels functionalized with β-cyclodextrin as a local paclitaxel delivery system.

J Biomed Mater Res A 2021 Dec 30;109(12):2625-2639. Epub 2021 Jun 30.

NOBIPOL, Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Trondheim, Norway.

Modification of drug delivery materials with beta-cyclodextrins (β-CyD) is known to increase solubility of poorly water-soluble drugs, protect drugs from degradation and sustain release. In this study, we developed a hydrogel drug delivery system for local paclitaxel delivery using the natural polysaccharide alginate functionalized with β-CyD-moieties. Paclitaxel was chosen due to its ability to form inclusion complexes with cyclodextrins. The rheological and mechanical properties of the prepared hydrogels were characterized, as well as in vitro release of the paclitaxel and in vitro activity on PC-3 prostate cancer cells. Introduction of β-CyD-moieties into the hydrogel reduces the mechanical properties of the gels compared to nonmodified gels. However, gelation kinetics were not markedly different. Furthermore, the β-CyD-modified alginate helped to reduce undesired crystallization of the paclitaxel in the gel and facilitated paclitaxel diffusion out of the gel network. Remarkably, the β-CyD grafted alginate showed increased capacity to complex paclitaxel compared to free HPβ-CyD. Release of both paclitaxel and degradation products were measured from the gels and were shown to have cytotoxic effects on the PC-3 cells. The results indicate that functionalized alginate with β-CyDs has potential as a material for drug delivery systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbm.a.37255DOI Listing
December 2021

Insights into the roles of charged residues in substrate binding and mode of action of mannuronan C-5 epimerase AlgE4.

Glycobiology 2021 Apr 1. Epub 2021 Apr 1.

Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Sem Sælands vei 6/8, N-7491 Trondheim, Norway.

Mannuronan C-5 epimerases catalyse the epimerization of monomer residues in the polysaccharide alginate, changing the physical properties of the biopolymer. The enzymes are utilized to tailor alginate to numerous biological functions by alginate-producing organisms. The underlying molecular mechanisms that control the processive movement of the epimerase along the substrate chain is still elusive. To study this, we have used an interdisciplinary approach combining molecular dynamics simulations with experimental methods from mutant studies of AlgE4, where initial epimerase activity and product formation were addressed with NMR spectroscopy, and characteristics of enzyme-substrate interactions were obtained with isothermal titration calorimetry and optical tweezers. Positive charges lining the substrate-binding groove of AlgE4 appear to control the initial binding of poly-mannuronate, and binding also seems to be mediated by both electrostatic and hydrophobic interactions. After the catalytic reaction, negatively charged enzyme residues might facilitate dissociation of alginate from the positive residues, working like electrostatic switches, allowing the substrate to translocate in the binding groove. Molecular simulations show translocation increments of two monosaccharide units before the next productive binding event resulting in MG-block formation, with the epimerase moving with its N-terminus towards the reducing end of the alginate chain. Our results indicate that the charge pair R343-D345 might be directly involved in conformational changes of a loop that can be important for binding and dissociation. The computational and experimental approaches used in this study complement each other, allowing for a better understanding of individual residues' roles in binding and movement along the alginate chains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/glycob/cwab025DOI Listing
April 2021

Protective Face Mask Filter Capable of Inactivating SARS-CoV-2, and Methicillin-Resistant and .

Polymers (Basel) 2021 Jan 8;13(2). Epub 2021 Jan 8.

Biomaterials and Bioengineering Lab, Centro de Investigación Traslacional San Alberto Magno, Universidad Católica de Valencia San Vicente Mártir, c/Guillem de Castro 94, 46001 Valencia, Spain.

Face masks have globally been accepted to be an effective protective tool to prevent bacterial and viral transmission, especially against indoor aerosol transmission. However, commercial face masks contain filters that are made of materials that are not capable of inactivating either SARS-CoV-2 or multidrug-resistant bacteria. Therefore, symptomatic and asymptomatic individuals can infect other people even if they wear them because some viable viral or bacterial loads can escape from the masks. Furthermore, viral or bacterial contact transmission can occur after touching the mask, which constitutes an increasing source of contaminated biological waste. Additionally, bacterial pathogens contribute to the SARS-CoV-2-mediated pneumonia disease complex, and their resistance to antibiotics in pneumonia treatment is increasing at an alarming rate. In this regard, herein, we report the development of a non-woven face mask filter fabricated with a biofunctional coating of benzalkonium chloride that is capable of inactivating more than 99% of SARS-CoV-2 particles in one minute of contact, and the life-threatening methicillin-resistant and (normalized antibacterial of 0.52 ± 0.04 and 0.72 ± 0.04, respectively). Nonetheless, despite the results obtained, further studies are needed to ensure the safety and correct use of this technology for the mass production and commercialization of this broad-spectrum antimicrobial face mask filter. Our novel protective non-woven face mask filter would be useful for many healthcare workers and researchers working in this urgent and challenging field.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/polym13020207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827663PMC
January 2021

Ion-exchange purification and structural characterization of five sulfated fucoidans from brown algae.

Glycobiology 2021 May;31(4):352-357

Max Planck Institute for Marine Microbiology, 28359 Bremen, Germany.

Fucoidans are a diverse class of sulfated polysaccharides integral to the cell wall of brown algae, and due to their various bioactivities, they are potential drugs. Standardized work with fucoidans is required for structure-function studies, but remains challenging since available fucoidan preparations are often contaminated with other algal compounds. Additionally, fucoidans are structurally diverse depending on species and season, urging the need for standardized purification protocols. Here, we use ion-exchange chromatography to purify different fucoidans and found a high structural diversity between fucoidans. Ion-exchange chromatography efficiently removes the polysaccharides alginate and laminarin and other contaminants such as proteins and phlorotannins across a broad range of fucoidans from major brown algal orders including Ectocarpales, Laminariales and Fucales. By monomer composition, linkage analysis and NMR characterization, we identified galacturonic acid, glucuronic acid and O-acetylation as new structural features of certain fucoidans and provided a novel structure of fucoidan from Durvillaea potatorum with α-1,3-linked fucose backbone and β-1,6 and β-1,3 galactose branches. This study emphasizes the use of standardized ion-exchange chromatography to obtain defined fucoidans for subsequent molecular studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/glycob/cwaa064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091464PMC
May 2021

A pair of esterases from a commensal gut bacterium remove acetylations from all positions on complex β-mannans.

Proc Natl Acad Sci U S A 2020 03 13;117(13):7122-7130. Epub 2020 Mar 13.

Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 1432 Ås, Norway;

β-mannans and xylans are important components of the plant cell wall and they are acetylated to be protected from degradation by glycoside hydrolases. β-mannans are widely present in human and animal diets as fiber from leguminous plants and as thickeners and stabilizers in processed foods. There are many fully characterized acetylxylan esterases (AcXEs); however, the enzymes deacetylating mannans are less understood. Here we present two carbohydrate esterases, CE2 and CE17, from the Firmicute , which together deacetylate complex galactoglucomannan (GGM). The three-dimensional (3D) structure of CE17 with a mannopentaose in the active site shows that the CBM35 domain of CE17 forms a confined complex, where the axially oriented C2-hydroxyl of a mannose residue points toward the Ser41 of the catalytic triad. Cavities on the CE17 surface may accept galactosylations at the C6 positions of mannose adjacent to the mannose residue being deacetylated (subsite -1 and +1). In-depth characterization of the two enzymes using time-resolved NMR, high-performance liquid chromatography (HPLC), and mass spectrometry demonstrates that they work in a complementary manner. CE17 exclusively removes the axially oriented 2--acetylations on any mannose residue in an oligosaccharide, including double acetylated mannoses, while the CE2 is active on 3-, 4-, and 6-acetylations. Activity of CE2 is dependent on CE17 removing 2--acetylations from double acetylated mannose. Furthermore, transacetylation of oligosaccharides with the 2--specific CE17 provided insight into how temperature and pH affects acetyl migration on manno-oligosaccharides.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1915376117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132267PMC
March 2020

Chitosan as a Wound Dressing Starting Material: Antimicrobial Properties and Mode of Action.

Int J Mol Sci 2019 Nov 24;20(23). Epub 2019 Nov 24.

Advanced Environmental Research Laboratories, Department of Biology-Chemistry, West University of Timisoara, Oituz 4, 300086 Timisoara, Romania.

Fighting bacterial resistance is one of the concerns in modern days, as antibiotics remain the main resource of bacterial control. Data shows that for every antibiotic developed, there is a microorganism that becomes resistant to it. Natural polymers, as the source of antibacterial agents, offer a new way to fight bacterial infection. The advantage over conventional synthetic antibiotics is that natural antimicrobial agents are biocompatible, non-toxic, and inexpensive. Chitosan is one of the natural polymers that represent a very promising source for the development of antimicrobial agents. In addition, chitosan is biodegradable, non-toxic, and most importantly, promotes wound healing, features that makes it suitable as a starting material for wound dressings. This paper reviews the antimicrobial properties of chitosan and describes the mechanisms of action toward microbial cells as well as the interactions with mammalian cells in terms of wound healing process. Finally, the applications of chitosan as a wound-dressing material are discussed along with the current status of chitosan-based wound dressings existing on the market.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms20235889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928789PMC
November 2019

Graphene oxide nanosheets versus carbon nanofibers: Enhancement of physical and biological properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) films for biomedical applications.

Int J Biol Macromol 2020 Jan 15;143:1000-1008. Epub 2019 Nov 15.

Facultad de Veterinaria y Ciencias Experimentales, Universidad Católica de Valencia San Vicente Mártir, c/Guillem de Castro 94, Valencia 46001, Spain. Electronic address:

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a microbial biodegradable polymer with a wide range of potential industrial applications. However, its biomedical uses could increase exponentially if certain physical and biological properties were enhanced without compromising on the non-cytotoxic property of this biocompatible polymer. Graphene oxide (GO) nanosheets and carbon nanofibers (CNFs) have proven to be very promising reinforcing agents for the development of new composite materials. Therefore, PHBV films were prepared with 1% w/w of GO nanosheets or CNFs with the aim of enhancing their compression performance, thermal behaviour, wettability and cell adhesion using canine adipose-derived mesenchymal stem cells, and antibacterial activity against the model bacterium Staphylococcus aureus. The results of this study showed that both nanomaterials produced similar enhancements of the physical properties. However, PHBV/GO exhibited higher proliferative activity against time, cell adhesion and antibacterial activity than that of PHBV/CNFs. Nonetheless, both PHBV/GO and PHBV/CNFs composite films have shown considerable promise for biomedical applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijbiomac.2019.10.034DOI Listing
January 2020

Molecular insight into a new low-affinity xylan binding module from the xylanolytic gut symbiont Roseburia intestinalis.

FEBS J 2020 05 20;287(10):2105-2117. Epub 2019 Nov 20.

Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark.

Efficient capture of glycans, the prime metabolic resources in the human gut, confers a key competitive advantage for gut microbiota members equipped with extracellular glycoside hydrolases (GHs) to target these substrates. The association of glycans to the bacterial cell surface is typically mediated by carbohydrate binding modules (CBMs). Here, we report the structure of RiCBM86 appended to a GH family 10 xylanase from Roseburia intestinalis. This CBM represents a new family of xylan binding CBMs present in xylanases from abundant and prevalent healthy human gut Clostridiales. RiCBM86 adopts a canonical β-sandwich fold, but shows structural divergence from known CBMs. The structure of RiCBM86 has been determined with a bound xylohexaose, which revealed an open and shallow binding site. RiCBM86 recognizes only a single xylosyl ring with direct hydrogen bonds. This mode of recognition is unprecedented amongst previously reported xylan binding type-B CBMs that display more extensive hydrogen-bonding patterns to their ligands or employ Ca to mediate ligand-binding. The architecture of RiCBM86 is consistent with an atypically low binding affinity (K about 0.5 mm for xylohexaose) compared to most xylan binding CBMs. Analyses using NMR spectroscopy corroborated the observations from the complex structure and the preference of RiCBM86 to arabinoxylan over glucuronoxylan, consistent with the largely negatively charged surface flanking the binding site. Mutational analysis and affinity electrophoresis established the importance of key binding residues, which are conserved in the family. This study provides novel insight into the structural features that shape low-affinity CBMs that mediate extended bacterial glycan capture in the human gut niche. DATABASES: Structural data are available in the protein data bank database under the accession number 6SGF. Sequence data are available in the GenBank database under the accession number EEV01588.1. The assignment of the Roseburia intestinalis xylan binding module into the CBM86 new family is available in the CAZy database (http://www.cazy.org/CBM86.html).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/febs.15117DOI Listing
May 2020

Graphene oxide in zinc alginate films: Antibacterial activity, cytotoxicity, zinc release, water sorption/diffusion, wettability and opacity.

PLoS One 2019 7;14(3):e0212819. Epub 2019 Mar 7.

Facultad de Veterinaria y Ciencias Experimentales, Universidad Católica de Valencia San Vicente Mártir, Valencia, Spain.

Alginate is considered an exceptional biomaterial due to its hydrophilicity, biocompatibility, biodegradability, nontoxicity and low-cost in comparison with other biopolymers. We have recently demonstrated that the incorporation of 1% graphene oxide (GO) into alginate films crosslinked with Ca2+ cations provides antibacterial activity against Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis, and no cytotoxicity for human keratinocyte HaCaT cells. However, many other reports in literature have shown controversial results about the toxicity of GO demanding further investigation. Furthermore, the synergic effect of GO with other divalent cations with intrinsic antibacterial and cytotoxic activity such as Zn2+ has not been explored yet. Thus, here, two commercially available sodium alginates were characterised and utilized in the synthesis of zinc alginate films with GO following the same chemical route reported for the calcium alginate/GO composites. The results of this study showed that zinc release, water sorption/diffusion and wettability depended significantly on the type of alginate utilized. Furthermore, Zn2+ and GO produced alginate films with increased water diffusion, wettability and opacity. However, neither the combination of GO with Zn2+ nor the use of different types of sodium alginates modified the antibacterial activity and cytotoxicity of the zinc alginates against these Gram-positive pathogens and human cells respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212819PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405205PMC
December 2019

Biosynthesis and Function of Long Guluronic Acid-Blocks in Alginate Produced by Azotobacter vinelandii.

Biomacromolecules 2019 04 19;20(4):1613-1622. Epub 2019 Mar 19.

Department of Biotechnology and Food Science , Norwegian University of Science and Technology, NTNU , Sem Sælands vei 6-8 , N-7491 Trondheim , Norway.

With the present accessibility of algal raw material, microbial alginates as a source for strong gelling material are evaluated as an alternative for advanced applications. Recently, we have shown that alginate from algal sources all contain a fraction of very long G-blocks (VLG), that is, consecutive sequences of guluronic acid (G) residues of more than 100 residues. By comparing the gelling properties of these materials with in vitro epimerized polymannuronic acid (poly-M) with shorter G-blocks, but comparable with the G-content, we could demonstrate that VLG have a large influence on gelling properties. Hypothesized to function as reinforcement bars, VLG prevents the contraction of the gels during formation (syneresis) and increases the Young's modulus (strength of the gel). Here we report that these VLG structures are also present in alginates from Azotobacter vinelandii and that these polymers consequently form stable, low syneretic gels with calcium, comparable in mechanical strength to algal alginates with the similar monomeric composition. The bacterium expresses seven different extracellular mannuronan epimerases (AlgE1-AlgE7), of which only the bifunctional epimerase AlgE1 seems to be able to generate the long G-blocks when acting on poly-M. The data implies evidence for a processive mode of action and the necessity of two catalytic sites to obtain the observed epimerization pattern. Furthermore, poly-M epimerized with AlgE1 in vitro form gels with comparable or higher rigidity and gel strength than gels made from brown seaweed alginate with matching G-content. These findings strengthen the viability of commercial alginate production from microbial sources.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biomac.8b01796DOI Listing
April 2019

Genome Mining of sp. YIM 130001 Isolated From Lichen Affords New Thiopeptide Antibiotic.

Front Microbiol 2018 19;9:3139. Epub 2018 Dec 19.

Department of Pharmacognosy, University of Vienna, Vienna, Austria.

bacteria are recognized as an important source for antibiotics with broad applications in human medicine and animal health. Here, we report the isolation of a new lichen-associating sp. YIM 130001 from the tropical rainforest in Xishuangbanna (Yunnan, China), which displayed antibacterial activity against . The draft genome sequence of this isolate strain revealed 18 putative biosynthetic gene clusters (BGCs) for secondary metabolites, which is an unusually low number compared to a typical streptomycete. Inactivation of a lantibiotic dehydrogenase-encoding gene from the BGC presumed to govern biosynthesis of a thiopeptide resulted in the loss of bioactivity. Using comparative HPLC analysis, two peaks in the chromatogram were identified in the extract from the wild-type strain, which were missing in the extract from the mutant. The compounds corresponding to the identified peaks were purified, and structure of one compound was elucidated using NMR. The compound, designated geninthiocin B, showed high similarity to several 35-membered macrocyclic thiopeptides geninthiocin, Val-geninthiocin and berninamycin A. Bioinformatics analysis of the geninthiocin B BGC revealed its close homology to that of berninamycins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2018.03139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6306032PMC
December 2018

H, C and N backbone and side-chain assignment of a carbohydrate binding module from a xylanase from Roseburia intestinalis.

Biomol NMR Assign 2019 04 22;13(1):55-58. Epub 2018 Sep 22.

NOBIPOL, Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Trondheim, Norway.

The N-terminal domain (residues 28-165) from the glycoside hydrolase family 10 from Roseburia intestinalis (RiCBMx), has been isotopically labeled and recombinantly expressed in Escherichia coli. Here we report H, C and N NMR chemical shift assignments for this carbohydrate binding module (CBM).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12104-018-9850-3DOI Listing
April 2019

Differential bacterial capture and transport preferences facilitate co-growth on dietary xylan in the human gut.

Nat Microbiol 2018 05 2;3(5):570-580. Epub 2018 Apr 2.

Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark.

Metabolism of dietary glycans is pivotal in shaping the human gut microbiota. However, the mechanisms that promote competition for glycans among gut commensals remain unclear. Roseburia intestinalis, an abundant butyrate-producing Firmicute, is a key degrader of the major dietary fibre xylan. Despite the association of this taxon to a healthy microbiota, insight is lacking into its glycan utilization machinery. Here, we investigate the apparatus that confers R. intestinalis growth on different xylans. R. intestinalis displays a large cell-attached modular xylanase that promotes multivalent and dynamic association to xylan via four xylan-binding modules. This xylanase operates in concert with an ATP-binding cassette transporter to mediate breakdown and selective internalization of xylan fragments. The transport protein of R. intestinalis prefers oligomers of 4-5 xylosyl units, whereas the counterpart from a model xylan-degrading Bacteroides commensal targets larger ligands. Although R. intestinalis and the Bacteroides competitor co-grew in a mixed culture on xylan, R. intestinalis dominated on the preferred transport substrate xylotetraose. These findings highlight the differentiation of capture and transport preferences as a possible strategy to facilitate co-growth on abundant dietary fibres and may offer a unique route to manipulate the microbiota based on glycan transport preferences in therapeutic interventions to boost distinct taxa.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-018-0132-8DOI Listing
May 2018

Human Chitotriosidase: Catalytic Domain or Carbohydrate Binding Module, Who's Leading HCHT's Biological Function.

Sci Rep 2017 06 5;7(1):2768. Epub 2017 Jun 5.

InBioS - Center for Protein Engineering, Department of Life Sciences, Université de Liège, Sart-Tilman, B4000, Liège, Belgium.

Chitin is an important structural component of numerous fungal pathogens and parasitic nematodes. The human macrophage chitotriosidase (HCHT) is a chitinase that hydrolyses glycosidic bonds between the N-acetyl-D-glucosamine units of this biopolymer. HCHT belongs to the Glycoside Hydrolase (GH) superfamily and contains a well-characterized catalytic domain appended to a chitin-binding domain (ChBD). Although its precise biological function remains unclear, HCHT has been described to be involved in innate immunity. In this study, the molecular basis for interaction with insoluble chitin as well as with soluble chito-oligosaccharides has been determined. The results suggest a new mechanism as a common binding mode for many Carbohydrate Binding Modules (CBMs). Furthermore, using a phylogenetic approach, we have analysed the modularity of HCHT and investigated the evolutionary paths of its catalytic and chitin binding domains. The phylogenetic analyses indicate that the ChBD domain dictates the biological function of HCHT and not its appended catalytic domain. This observation may also be a general feature of GHs. Altogether, our data have led us to postulate and discuss that HCHT acts as an immune catalyser.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-02382-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5459812PMC
June 2017

Interactions of a fungal lytic polysaccharide monooxygenase with β-glucan substrates and cellobiose dehydrogenase.

Proc Natl Acad Sci U S A 2016 May 5;113(21):5922-7. Epub 2016 May 5.

Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway;

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched β-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O2 (-) Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme-substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1602566113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889390PMC
May 2016

Energy Landscape of Alginate-Epimerase Interactions Assessed by Optical Tweezers and Atomic Force Microscopy.

PLoS One 2015 23;10(10):e0141237. Epub 2015 Oct 23.

NOBIPOL, Dept. of Biotechnology, Norwegian University of Science and Technology, NO-7491, Trondheim, Norway.

Mannuronan C-5 epimerases are a family of enzymes that catalyze epimerization of alginates at the polymer level. This group of enzymes thus enables the tailor-making of various alginate residue sequences to attain various functional properties, e.g. viscosity, gelation and ion binding. Here, the interactions between epimerases AlgE4 and AlgE6 and alginate substrates as well as epimerization products were determined. The interactions of the various epimerase-polysaccharide pairs were determined over an extended range of force loading rates by the combined use of optical tweezers and atomic force microscopy. When studying systems that in nature are not subjected to external forces the access to observations obtained at low loading rates, as provided by optical tweezers, is a great advantage since the low loading rate region for these systems reflect the properties of the rate limiting energy barrier. The AlgE epimerases have a modular structure comprising both A and R modules, and the role of each of these modules in the epimerization process were examined through studies of the A- module of AlgE6, AlgE6A. Dynamic strength spectra obtained through combination of atomic force microscopy and the optical tweezers revealed the existence of two energy barriers in the alginate-epimerase complexes, of which one was not revealed in previous AFM based studies of these complexes. Furthermore, based on these spectra estimates of the locations of energy transition states (xβ), lifetimes in the absence of external perturbation (τ0) and free energies (ΔG#) were determined for the different epimerase-alginate complexes. This is the first determination of ΔG# for these complexes. The values determined were up to 8 kBT for the outer barrier, and smaller values for the inner barriers. The size of the free energies determined are consistent with the interpretation that the enzyme and substrate are thus not tightly locked at all times but are able to relocate. Together with the observed different affinities determined for AlgE4-polymannuronic acid (poly-M) and AlgE4-polyalternating alginate (poly-MG) macromolecular pairs these data give important contribution to the growing understanding of the mechanisms underlying the processive mode of these enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141237PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4619708PMC
June 2016

Heparin-like properties of sulfated alginates with defined sequences and sulfation degrees.

Biomacromolecules 2014 Jul 20;15(7):2744-50. Epub 2014 Jun 20.

Department of Biotechnology, Norwegian University of Science and Technology , Sem Sælands vei 6/8, 7034 Trondheim, Norway.

Sulfated glycosaminoglycans have a vast range of protein interactions relevant to the development of new biomaterials and pharmaceuticals, but their characterization and application is complicated mainly due to a high structural variability and the relative difficulty to isolate large quantities of structurally homogeneous samples. Functional and versatile analogues of heparin/heparan sulfate can potentially be created from sulfated alginates, which offer structure customizability through targeted enzymatic epimerization and precise tuning of the sulfation degree. Alginates are linear polysaccharides consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G), derived from brown algae and certain bacteria. The M/G ratio and distribution of blocks are critical parameters for the physical properties of alginates and can be modified in vitro using mannuronic-C5-epimerases to introduce sequence patterns not found in nature. Alginates with homogeneous sequences (poly-M, poly-MG, and poly-G) and similar molecular weights were chemically sulfated and structurally characterized by the use of NMR and elemental analysis. These sulfated alginates were shown to bind and displace HGF from the surface of myeloma cells in a manner similar to heparin. We observed dependence on the sulfation degree (DS) as well as variation in efficacy based on the alginate monosaccharide sequence, relating to relative flexibility and charge density in the polysaccharide chains. Co-incubation with human plasma showed complement compatibility of the alginates and lowering of soluble terminal complement complex levels by sulfated alginates. The sulfated polyalternating (poly-MG) alginate proved to be the most reproducible in terms of precise sulfation degrees and showed the greatest relative degree of complement inhibition and HGF interaction, maintaining high activity at low DS values.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bm500602wDOI Listing
July 2014

RGD-peptide modified alginate by a chemoenzymatic strategy for tissue engineering applications.

J Biomed Mater Res A 2015 Mar 28;103(3):896-906. Epub 2014 May 28.

MI Lab and Department of Circulation and Medical Imaging, Norwegian University of Science and Technology, Trondheim, Norway.

One of the main challenges in tissue engineering and regenerative medicine is the ability to maintain optimal cell function and survival post-transplantation. Biomaterials such as alginates are commonly used for immunoisolation, while they may also provide structural support to the cell transplants by mimicking the extracellular matrix. In this study, arginine-glycine-aspartate (RGD)-peptide-coupled alginates of tailored composition were produced by adopting a unique chemoenzymatic strategy for substituting the nongelling mannuronic acid on the alginate. Alginates with and without RGD were produced with high and low content of G. Using carbodiimide chemistry 0.1-0.2% of the sugar units were substituted by peptide. Furthermore, the characterization by (1)H-nuclear magnetic resonance (NMR) revealed by-products from the coupling reaction that partly could be removed by coal filtration. Olfactory ensheathing cells (OECs) and myoblasts were grown in two-dimensional (2D) and 3D cultures of RGD-peptide modified or unmodified alginates obtained by the chemoenzymatically strategy and compared to native alginate. Both OECs and myoblasts adhered to the RGD-peptide modified alginates in 2D cultures, forming bipolar protrusions. OEC encapsulation resulted in cell survival for up to 9 days, thus demonstrating the potential for short-term 3D culture. Myoblasts showed long-term survival in 3D cultures, that is, up to 41 days post encapsulation. The RGD modifications did not result in marked changes in cell viability in 3D cultures. We demonstrate herein a unique technique for tailoring peptide substituted alginates with a precise and flexible composition, conserving the gel forming properties relevant for the use of alginate in tissue engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbm.a.35230DOI Listing
March 2015

Methodologies to increase the transformation efficiencies and the range of bacteria that can be transformed.

Appl Microbiol Biotechnol 2010 Feb 28;85(5):1301-13. Epub 2009 Nov 28.

Vectron Biosolutions AS, Pirsenteret, 7462 Trondheim, Norway.

Methods for transfer of exogenous DNA into cells are essential for genetics and molecular biology, and the lack of effective methods hampers research on many different species of bacteria which have shown to be particularly recalcitrant to transformation. This review presents the progress on the development of methods for artificial transformation of bacteria with emphasis on different methodologies and the range of bacteria that can be transformed. The methods' strengths and weaknesses are described.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00253-009-2349-1DOI Listing
February 2010

Use of cyclodextrin and its derivatives for increased transformation efficiency of competent bacterial cells.

Appl Microbiol Biotechnol 2009 Jun 28;83(3):589-96. Epub 2009 Apr 28.

NOBIPOL, Department of Biotechnology, Norwegian University of Science and Technology, Sem Saelands vei 6/8, 7491 Trondheim, Norway.

Methodologies for introduction of DNA into cells are essential in molecular genetics and vital for applications such as genetic engineering and gene therapy. The use of cyclodextrins (CyDs) for increased efficiency of introducing DNA into eukaryotic cells (transfection) has been reported, but CyDs' effect on the introduction of DNA into bacterial cells (transformation) is unknown. Here, we have investigated the potential of using CyDs in the transformation of chemically competent in-house, commercially available, and, on non-competent bacterial cells, with plasmid DNA of two different sizes. Possible interactions between CyDs and DNA were studied with nuclear magnetic resonance (NMR) spectroscopy. The presence of CyDs resulted in an up to fourfold increment of the transformation rate for in-house cells, with beta-CyD and derivates giving the strongest effect. For commercial cells and transformation with megaplasmids, a more moderate effect around 1.4-fold was obtained. However, CyDs have little or no effect on DNA uptake by noncompetent cells. Results obtained from NMR spectroscopy show no interactions between CyDs and DNA-like molecules, which indicated that the CyDs' effect is related to the bacterial cell wall.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00253-009-1907-xDOI Listing
June 2009

Preparation of high purity monodisperse oligosaccharides derived from mannuronan by size-exclusion chromatography followed by semi-preparative high-performance anion-exchange chromatography with pulsed amperometric detection.

Carbohydr Res 2009 Jan 30;344(2):255-9. Epub 2008 Oct 30.

NOBIPOL, Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.

Oligosaccharides of ([4)-beta-d-ManpA-(-->](n)) with a degree of polymerisation (DP) of 5, 10 and 15 were generated by partial acid hydrolysis of alginate mannuronan. These were subsequently purified by a combination of size-exclusion chromatography and semi-preparative high-performance anion-exchange chromatography with pulsed amperometric detection. The purity of the isolated oligosaccharides was greater than 96%. With automated operation of the chromatography system, milligram quantities can be generated over a period of a few days. Thus, our methodology now offers some significant advantages over earlier, including our own, protocols focused on uronic acid oligomers, where the final products are either not as pure or more starting material is needed to generate an equivalent yield of product. Removal of ammonium ions in collected fractions after size-exclusion chromatography and prior to freeze-drying was found to be essential to prevent the formation of imines and subsequent Maillard browning products.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.carres.2008.10.022DOI Listing
January 2009
-->