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1.	Clifton, L. A., et al. (författare) Characterizing biomaterial complexity 2009 Ingår i: Materials Today. - 1369-7021. ; 12:7-8, s. 86-91 Tidskriftsartikel (refereegranskat)abstract Biomaterials research will always require a range of techniques to examine structure and function on a range of length scales and in a range of settings. Neutron scattering provides a unique way of disentangling the molecular and structural complexity of biomaterials through study of the constituent components. We examine how the technique has been used to study surface immobilized proteins and lipid films, floating lipid bilayers as mimics of in vitro planar membranes, and formation of fibres from solution by insects and spiders.
2.	Del Giudice, Alessandra, et al. (författare) Structural response of human serum albumin to oxidation : Biological buffer to local formation of hypochlorite 2016 Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 120:40, s. 12261-12271 Tidskriftsartikel (refereegranskat)abstract The most abundant plasma protein, human serum albumin (HSA), plays a key part in the body's antioxidant defense against reactive species. This study was aimed at correlating oxidant-induced chemical and structural effects on HSA. Despite the chemical modification induced by the oxidant hypochlorite, the native shape is preserved up to oxidant/HSA molar ratio <80, above which a structural transition occurs in the critical range 80-120. This conformational variation involves the drifting of one of the end-domains from the rest of the protein and corresponds to the loss of one-third of the α-helix and a net increase of the protein negative charge. The transition is highly reproducible suggesting that it represents a well-defined structural response typical of this multidomain protein. The ability to tolerate high levels of chemical modification in a folded or only partially unfolded state, as well as the stability to aggregation, provides albumin with optimal features as a biological buffer for the local formation of oxidants. (Graph Presented).
3.	Del Giudice, Alessandra, et al. (författare) The effect of fatty acid binding in the acid isomerizations of albumin investigated with a continuous acidification method 2018 Ingår i: Colloids and Surfaces B: Biointerfaces. - : Elsevier BV. - 0927-7765. ; 168, s. 109-116 Tidskriftsartikel (refereegranskat)abstract The protein Human Serum Albumin (HSA) is known to undergo conformational transitions towards partially unfolded forms triggered by acidification below pH 4.5. The extent of Fatty Acids (FA) binding has been thought to have an impact on the conformational equilibrium between the native and acid forms and to be a possible explanation for the observation of more than one band in early electrophoretic migration experiments at pH 4. We compared the acid-induced unfolding processes of commercial FA-free HSA, commercial “fatted” HSA and FA-HSA complexes, prepared at FA:HSA molar ratios between 1 and 6 by simple mixing and equilibration. We used a method for continuous acidification based on the hydrolysis of glucono-δ-lactone from pH 7 to pH 2.5, and followed the average protein changes by the blue shift of the intrinsic fluorescence emission and by performing a small angle X-ray scattering analysis on selected samples. The method also allowed for continuous monitoring of the increase of turbidity and laser light scattering of the protein samples related to the release of the insoluble ligands with acidification. Our results showed that the presence of FA interacting with albumin, an aspect often neglected in biophysical studies, affects the conformational response of the protein to acidification, and slightly shifts the loss of the native shape from pH 4.2 to pH 3.6. This effect increased with the FA:HSA molar ratio so that with three molar equivalents a saturation was reached, in agreement with the number of high-affinity binding sites reported for the FA. These findings confirm that a non-uniform level of ligand binding in an albumin sample can be an explanation for the early-observed conformational heterogeneity at pH 4.
4.	Del Giudice, Alessandra, et al. (författare) Time-Dependent pH Scanning of the Acid-Induced Unfolding of Human Serum Albumin Reveals Stabilization of the Native Form by Palmitic Acid Binding 2017 Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 121:17, s. 4388-4399 Tidskriftsartikel (refereegranskat)abstract The most abundant plasma protein, human serum albumin (HSA), is known to undergo several conformational transitions in an acidic environment. To avoid buffer effects and correlate global and local structural changes, we developed a continuous acidification method and simultaneously monitored the protein changes by both small-angle scattering (SAXS) and fluorescence. The progressive acidification, based on the hydrolysis of glucono-δ-lactone from pH 7 to pH 2.5, highlighted a multistep unfolding involving the putative F form (pH 4) and an extended and flexible conformation (pH < 3.5). The scattering profile of the F form was extracted by component analysis and further 3D modeled. The effect of acid unfolding at this intermediate stage was assigned to the rearrangement of the three albumin domains drifting apart toward a more elongated conformation, with a partial unfolding of one of the outer domains. To test the stabilizing effect of fatty acids, here palmitic acid, we compared the acid unfolding process of albumin with and without ligand. We found that when binding the ligand, the native conformation was favored up to lower pH values. Our approach solved the problem of realizing a continuous, homogeneous, and tunable acidification with simultaneous characterization applicable to study processes triggered by a pH decrease.
5.	Dicko, Cedric, et al. (författare) Differential scanning fluorimetry illuminates silk feedstock stability and processability. 2016 Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 12:1, s. 255-262 Tidskriftsartikel (refereegranskat)abstract The ability to design and implement silk feedstock formulations for tailored spinning has so far eluded the bioengineers. Recently, the high throughput screening technique of differential scanning fluorimetry (DSF) demonstrated the link between the instability transition temperature (Ti) and the processability of the silk feedstock. Using DSF we screened a large set of chemicals known to affect solvent quality. A multivariate analysis of the results shows that, regardless of the diversity of chemicals, three groupings are significantly distinguishable: G1 = similar to native silk; G2 = largely dominated by electrostatic interactions; and G3 = dominated by chelating interactions. We propose a thermodynamic analysis based on a pre- and post-transition fit to estimate the van't Hoff enthalpies (ΔHv) and the instability temperature (Ti). Our analysis shows that the ΔTi and ΔHv values were distinct: G1 (ΔTi = 0.23 ± 0.2; ΔHv = -159.1 ± 5.6 kcal mol(-1)), G2 (ΔTi = -7.3 ± 0.7; ΔHv = -191.4 ± 5.5 kcal mol(-1)), and G3 (ΔTi = -19.9 ± 3.3; ΔHv = -68.8 ± 6.0 kcal mol(-1)). Our analysis further combined the ΔTi value and the ΔHv value using stability ΔΔG to find that G1 only marginally stabilizes native silks (ΔΔG = -0.15 ± 0.04 kcal mol(-1)), whereas G2 and G3 destabilize native silk (ΔΔG = 3.8 ± 0.11 and ΔΔG = 3.8 ± 0.3 kcal mol(-1), respectively). Here our analysis shows that native silk has a complex multistep transition that is possibly non-cooperative. However, all three groupings also show a direct and cooperative transition with varied stabilization effects. This analysis suggests that native silks are able to sample multiple substates prior to undergoing (or to delay) the final transition. We conclude by hypothesizing that the observed energetic plasticity may be mediated by a fragile packaging of the silk tertiary structure that is readily lost when the solvent quality changes.
6.	Dicko, Cedric, et al. (författare) NUrF-Optimization of in situ UV-vis and fluorescence and autonomous characterization techniques with small-angle neutron scattering instrumentation 2020 Ingår i: Review of Scientific Instruments. - : AMER INST PHYSICS. - 0034-6748 .- 1089-7623. ; 91:7 Tidskriftsartikel (refereegranskat)abstract We have designed, built, and validated a (quasi)-simultaneous measurement platform called NUrF, which consists of neutron small-angle scattering, UV-visible, fluorescence, and densitometry techniques. In this contribution, we illustrate the concept and benefits of the NUrF setup combined with high-performance liquid chromatography pumps to automate the preparation and measurement of a mixture series of Brij35 nonionic surfactants with perfluorononanoic acid in the presence of a reporter fluorophore (pyrene).
7.	Ferreira, Betina M.P., et al. (författare) 3D Structure and Mechanics of Silk Sponge Scaffolds Is Governed by Larger Pore Sizes 2020 Ingår i: Frontiers in Materials. - : Frontiers Media SA. - 2296-8016. ; 7 Tidskriftsartikel (refereegranskat)abstract Three-dimensional scaffolds play an essential role in tissue engineering. Although essential, the tunability of the 3D scaffolds mechanical and transport properties remains a challenge. In this work, we present new approaches to advance the field. First, we applied our progressive pH acidification to mimic the natural silk gelation process before ice-templating (−20 and −80°C); second, we fitted the mechanical properties using a connectivity model; third, we fitted the scaffolds mechanical relaxation to understand the transport properties; and fourth we used micro-CT to correlate the process parameters to the scaffolds' performances. Our results suggested that the free shrinkage of the scaffolds determined their final properties. We found, however, that the porosity (above 90%) was anisotropic, similarly the tortuosity (between 1 and 1.3). We identified two major pore dimensions, the first one between 10 and 20 μm, and the second between 50 and 130 μm. Mechanically, our model suggested that the bulk modulus captured the elastic contribution and was controlled predominantly by the silk concentration. We tentatively associated the fractional modulus 1 to the collapse of the larger pores structures and was controlled mostly by the process temperature. We assigned the slow relaxation to the transport of fluid in the silk sponge scaffolds; and the fast relaxation with a viscoelastic relaxation. The silk concentration and process temperatures did not influence the latter. Overall, our use of the tomography, mechanical test, and detailed statistical analysis provides inroads into the interplay between process parameters (silk concentration and process temperature) and the multiple responses of the silk sponge scaffolds. The development of a new mechanical fitting for the compression test helped capture simply the different failure modes in the sponge scaffolds as well as correlating those events to relaxation and eventually transport properties.
8.	Gilbert, Jennifer, et al. (författare) Immobilisation of β-galactosidase within a lipid sponge phase: structure, stability and kinetics characterisation 2019 Ingår i: Nanoscale. - 2040-3372. ; 11:44, s. 21291-21301 Tidskriftsartikel (refereegranskat)abstract In the formulation of an active enzyme enclosed in a matrix for controlled delivery, it is a challenge to achieve a high protein load and to ensure high activity of the protein. For the first time to our knowledge, we report the use of a highly swollen lipid sponge (L3) phase for encapsulation of the large active enzyme, β-galactosidase (β-gal, 238 kDa). This enzyme has large relevance for applications in, e.g. the production of lactose free milk products. The formulation consisted of diglycerol monooleate (DGMO), and a mixture of mono-, di- and triglycerides (Capmul GMO-50) stabilised by polysorbate 80 (P80). The advantage of this type of matrix is that it can be produced on a large scale with a fairly simple and mild process as the system is in practice self-dispersing, yet it has a well-defined internal nano-structure. Minor effects on the sponge phase structure due to the inclusion of the enzyme were observed using small angle X-ray scattering (SAXS). The effect of encapsulation on the enzymatic activity and kinetic characteristics of β-galactosidase activity was also investigated and can be related to the enzyme stability and confinement within the lipid matrix. The encapsulated β-galactosidase maintained its activity for a significantly longer time when compared to the free solution at the same temperature. Differences in the particle size and charge of sponge-like nanoparticles (L3-NPs) with and without the enzyme were analysed by dynamic light scattering (DLS) and zeta-potential measurements. Moreover, all the initial β-galactosidase was encapsulated within L3-NPs as revealed by size exclusion chromatography.
9.	Gong, Haiyue, et al. (författare) Ag−Polymer nanocomposites for capture, detection, and destruction of bacteria 2019 Ingår i: ACS Applied Nano Materials. - : American Chemical Society (ACS). - 2574-0970. ; 2, s. 1655-1663 Tidskriftsartikel (refereegranskat)abstract Bacterial infection is one of the major problems for human health. To prevent outbreak of bacteria-caused diseases, early diagnosis of bacterial pathogen and effective destruction of pathogenic microorganisms are in urgent need. In this work, we developed a new multifunctional nanocomposite material that can effectively capture and destroy bacteria. Epoxide-modified nanoparticles were synthesized by microemulsion polymerization and precipitation polymerization. The epoxide groups on the particle surface were reacted with polyethylenimine to introduce cationic amine groups. The amine groups on the nanoparticle surface enhanced the colloidal stability of the particles’ suspension and provided multivalent interactions to bind and destroy the bacteria. After further modification with Ag nanoparticles, the final composite nanomaterial was able to not only capture and destroy Gram-negative bacteria but also allow the bacteria’s fingerprint spectra to be obtained through surface-enhanced Raman scattering.The multifunctional nanoparticles developed in this work offer a new approach toward fast capture, detection, and destruction of pathogenic bacteria.
10.	Greving, Imke, et al. (författare) Structural Diversity of Native Major Ampullate, Minor Ampullate, Cylindriform, and Flagelliform Silk Proteins in Solution 2020 Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1526-4602 .- 1525-7797. ; 21:8, s. 3387-3393 Tidskriftsartikel (refereegranskat)abstract The foundations of silk spinning, the structure, storage, and activation of silk proteins, remain highly debated. By combining solution small-angle neutron and X-ray scattering (SANS and SAXS) alongside circular dichroism (CD), we reveal a shape anisotropy of the four principal native spider silk feedstocks from Nephila edulis. We show that these proteins behave in solution like elongated semiflexible polymers with locally rigid sections. We demonstrated that minor ampullate and cylindriform proteins adopt a monomeric conformation, while major ampullate and flagelliform proteins have a preference for dimerization. From an evolutionary perspective, we propose that such dimerization arose to help the processing of disordered silk proteins. Collectively, our results provide insights into the molecular-scale processing of silk, uncovering a degree of evolutionary convergence in protein structures and chemistry that supports the macroscale micellar/pseudo liquid crystalline spinning mechanisms proposed by the community.
11.	Haas, Sylvio, et al. (författare) Combined SAXS/UV-vis/Raman as a Diagnostic and Structure Resolving Tool in Materials and Life Sciences Applications. 2014 Ingår i: The Journal of Physical Chemistry Part B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 118:8, s. 2264-2273 Tidskriftsartikel (refereegranskat)abstract In order to diagnose and fully correlate structural, chemical, and functional features of macromolecules and particles in solution, we propose the integration of spectroscopy and scattering on the same measuring volume and at the same time in a dedicated sample environment with multiple probes. Combined SAXS/UV-vis and SAXS/Raman information are employed to study the radiation damage effect in proteins in solution and the scattering from single wall carbon nanotubes (SWNTs) in SDS dispersion, respectively. In the first case, a clear correlation is observed between the time dependence of the radius of gyration (Rg) of the protein determined by SAXS and the turbidity of the protein solution extracted from simultaneous UV-vis measurements. In the second case, the ratio of bundled/isolated carbon nanotubes is obtained unambiguously through proper modeling of the scattering data and cross-validated with the Raman information. The uses of convex constraint analysis (CCA) and two-dimensional correlation analyses (2DCOS and 2DHCOS) are introduced to fully explore the combination of data sets from different techniques and to extract unique insights from the sample.
12.	Hajizadeh, Solmaz, et al. (författare) Interaction of haemin with albumin-based macroporous cryogel : Adsorption isotherm and fluorescence quenching studies 2022 Ingår i: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media SA. - 2296-4185. ; 10 Tidskriftsartikel (refereegranskat)abstract Albumin-based cryogels for capturing haemin were synthesised by crosslinking different biomolecules, bovine serum albumin (BSA) and ovalbumin (OVA). The impact of the protein and coupling agent concentrations on cryogel’s mechanical properties, swelling ratios and polymerisation yields, as well as autoclaving as a post-treatment on the cryogel, were studied. We found that BSA (50 mg/ml) and the crosslinker (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, 46 mg/ml) formed a cryogel with optimum physical characteristics at a comparatively low protein concentration. The cryogel’s mechanical stability was increased using a double-layer cryogel approach by crosslinking the BSA proteins at subzero temperature inside an acrylamide and hydroxyethyl methacrylate premade cryogels. Batch binding and kinetic adsorption isotherms of haemin on the cryogels were assessed to evaluate their binding capacity toward the porphyrin molecule. The results showed that single-layer cryogels (BSA and OVA) had a higher capacity (∼0.68 mg/ml gel) and higher reaction rate constant towards haemin adsorption than double-layer gels. In contrast, the double-layer cryogels had higher mechanical strength than single-layer gels. The experimental results suggested that the cryogels followed the Freundlich model and the pseudo-second-order isotherm for batch adsorption and kinetics, respectively. The interaction between haemin and the gels was studied by fluorescence quenching. We found between 1.1 and 1.6 binding sites for different cryogels.
13.	Holland, Chris, et al. (författare) Distinct structural and optical regimes in natural silk spinning 2012 Ingår i: Biopolymers. - : Wiley. - 0006-3525. ; 97:6, s. 368-373 Tidskriftsartikel (refereegranskat)abstract This study investigates the relationship between birefringence and mechanical properties in the dragline silk of the gold orb weaving spider Nephila edulis. Using a custom birefringence-tensile testing device, we probed the orientation and water-induced swelling of fibers spun at variety of drawing rates ranging from 0.003 to 400 mm s-1. Our results indicate that based upon drawing rate, silk fibers fall into three distinct regimes each with characteristic orientation and swelling properties. Further investigation using in situ tensile testing reveals interactions between a fiber's drawing speed, mechanical properties, and orientation that support previous model predictions. We propose that simultaneous birefringence-tensile testing provides a unique and readily accessible insight into the structural behavior of this interesting and important biomaterial. (C) 2011 Wiley Periodicals, Inc. Biopolymers 97: 368373, 2012.
14.	Horrocks, Nicholas P. C., et al. (författare) The silkmoth cocoon as humidity trap and waterproof barrier 2013 Ingår i: Comparative Biochemistry and Physiology A. - : Elsevier BV. - 1531-4332. ; 164:4, s. 645-652 Tidskriftsartikel (refereegranskat)abstract To better understand how silkmoth cocoons maintain the correct internal moisture levels for successful pupation, we examined cocoons from the long-domesticated mulberry silkmoth Bombyx mori as well as from two wild silkmoth species, Antheraea pernyi and Philosamia cynthia ricini. We determined fluid-independent values for the porosity, tortuosity and permeability of the inner and outer surfaces of cocoons. Permeabilities were low and, with the exception of A. pernyi cocoons, inner surfaces were less permeable than outer surfaces. B. mori cocoons exhibited the highest permeability overall, but only at the outer surface, while A. pernyi cocoons appeared to show different patterns from the other species tested. We discuss our findings in light of the ecophysiology of the various species and propose a 'tortuous path' model to help explain our results. The model describes how the structure of the inner and outer layers of the cocoon allows it to function as both a humidity trap and a waterproof barrier, providing optimum conditions for the successful development of the pupa. (C) 2013 Elsevier Inc. All rights reserved.
15.	Humphreys, Ben A., et al. (författare) The Influence of pH on the Lipase Digestion of Nanosized Triolein, Diolein and Monoolein films 2022 Ingår i: Frontiers in Soft Matter. - : Frontiers Media SA. - 2813-0499. ; 2 Tidskriftsartikel (refereegranskat)abstract Herein we studied the processes at the liquid aqueous interface at pH 7 and 8.5 during Thermomyces lanuginosus lipase-catalyzed hydrolysis of nanosized tri-, di- and mono-olein films deposited on a planar substrate. By employing a combination of ellipsometry, QCM-D and ATR-FTIR, we were able to reveal the physical properties of the thin films at high time resolution throughout the initial hydration and subsequent digestion, as well as the main chemical species present before and after lipolysis. The ATR-FTIR results showed that the degree of digestion and protonated state of the oleic acid produced in the reaction are highly dependent on the pH of the aqueous solvent. Furthermore, the ellipsometry and QCM-D results reveal that the duration of the lag phase observed before lipolysis was detected and the magnitude and type of changes to the physical properties of the thin films throughout digestion was influenced by whether the initial substrate consisted of tri-, di- or mono-olein.
16.	Kettisen, Karin, et al. (författare) Site-Specific Introduction of Negative Charges on the Protein Surface for Improving Global Functions of Recombinant Fetal Hemoglobin 2021 Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8 Tidskriftsartikel (refereegranskat)abstract Due to its compatible oxygen-transporting abilities, hemoglobin (Hb) is a protein of interest in the development of artificial oxygen therapeutics. Despite continuous formulation attempts, extracellular Hb solution often exhibits undesirable reactions when applied in vivo. Therefore, protein engineering is frequently used to examine alternative ways of controlling the unwanted reactions linked to cell-free Hb solutions. In this study, three mutants of human fetal hemoglobin (HbF) are evaluated; single mutants αA12D and αA19D, and a double mutant αA12D/A19D. These variants were obtained by site-directed mutagenesis and recombinant production in E. coli, and carry negative charges on the surface of the α-subunit at the designated mutation sites. Through characterization of the mutant proteins, we found that the substitutions affected the protein in several ways. As expected, the isoelectric points (pIs) were lowered, from 7.1 (wild-type) down to 6.6 (double mutant), which influenced the anion exchange chromatographic procedures by shifting conditions toward higher conductivity for protein elution. The biological and physiological properties of HbF could be improved by these small modifications on the protein surface. The DNA cleavage rate associated with native HbF could be reduced by 55%. In addition, the negatively charged HbF mutant had an extended circulation time when examined in a mouse model using top load Hb additions. At the same time, the mutations did not affect the overall structural integrity of the HbF molecule, as determined by small-angle X-ray scattering. In combination with circular dichroism and thermal stability, modest structural shifts imposed by the mutations could possibly be related to changes in secondary structure or reorganization. Such local deformations were too minor to be determined within the resolution of the structural data; and overall, unchanged oxidation and heme loss kinetics support the conclusion that the mutations did not adversely affect the basic structural properties of Hb. We confirm the value of adding negatively charged residues onto the surface of the protein to improve the global functions of recombinant Hb.
17.	Kronenberger, Katrin, et al. (författare) A novel marine silk 2012 Ingår i: Die Naturwissenschaften. - : Springer Science and Business Media LLC. - 1432-1904. ; 99:1, s. 3-10 Tidskriftsartikel (refereegranskat)abstract The discovery of a novel silk production system in a marine amphipod provides insights into the wider potential of natural silks. The tube-building corophioid amphipod Crassicorophium bonellii produces from its legs fibrous, adhesive underwater threads that combine barnacle cement biology with aspects of spider silk thread extrusion spinning. We characterised the filamentous silk as a mixture of mucopolysaccharides and protein deriving from glands representing two distinct types. The carbohydrate and protein silk secretion is dominated by complex β-sheet structures and a high content of charged amino acid residues. The filamentous secretion product exits the gland through a pore near the tip of the secretory leg after having moved through a duct, which subdivides into several small ductules all terminating in a spindle-shaped chamber. This chamber communicates with the exterior and may be considered the silk reservoir and processing/mixing space, in which the silk is mechanically and potentially chemically altered and becomes fibrous. We assert that further study of this probably independently evolved, marine arthropod silk processing and secretion system can provide not only important insights into the more complex arachnid and insect silks but also into crustacean adhesion cements.
18.	Kurut, Anil, et al. (författare) Dimerization of Terminal Domains in Spiders Silk Proteins Is Controlled by Electrostatic Anisotropy and Modulated by Hydrophobic Patches 2015 Ingår i: ACS Biomaterials Science & Engineering. - : American Chemical Society (ACS). - 2373-9878. ; 1:6, s. 363-371 Tidskriftsartikel (refereegranskat)abstract The well-tuned spinning technology from spiders has attracted many researchers with the promise of producing high-performance, biocompatible, and yet biodegradable fibers. So far, the intricate chemistry and rheology of spinning have eluded us. A breakthrough was achieved recently, when the 3D structures of the N and C terminal domains of spider dragline silk were resolved and their pH-induced dimerization was revealed. To understand the terminal domains' dimerization mechanisms, we developed a protein model based on the experimental structures that reproduces charge and hydrophobic anisotropy of the complex protein surfaces. Monte Carlo simulations were used to study the thermodynamic dimerization of the N-terminal domain as a function of pH and ionic strength. We show that the hydrophobic and electrostatic anisotropies of the N-terminal domain cooperate constructively in the association process. The dipolar attractions at pH 6 lead to weakly bound dimers by forcing an antiparallel monomer orientation, stabilized by hydrophobic locking at close separations. Elevated salt concentrations reduce the thermodynamic dimerization constant due to screened electrostatic dipolar attraction. Moreover, the mutations on ionizable residues reveal a free energy of binding, proportional to the dipole moment of the mutants. It has previously been shown that dimers, formed at pH 6, completely dissociate at pH 7, which is thought to be due to altered protein charges. In contrast, our study indicates that the pH increase has no influence on the charge distribution of the N-terminal domain. Instead, the pH-induced dissociation is due to an adapted, loose conformation at pH 7, which significantly hampers both electrostatic and hydrophobic attractive interactions.
19.	Ouédraogo, Jean Claude W., et al. (författare) Enhanced extraction of flavonoids from Odontonema strictum leaves with antioxidant activity using supercritical carbon dioxide fluid combined with ethanol 2018 Ingår i: Journal of Supercritical Fluids. - : Elsevier BV. - 0896-8446. ; 131, s. 66-71 Tidskriftsartikel (refereegranskat)abstract Flavonoids were extracted from Odontonema strictum leaves by supercritical carbon dioxide, with ethanol. The effect of three independents variables (time, pressure and temperature) on the Total Flavonoid Content (TFC) were optimized by factorial investigation. The TFC and flavonoid recovery varies respectively from 99.33 to 247.78 mg/g of dried extract and 10.68–18.92 mg/g of dried leaves powder, while the conventional solvent extraction (CSE) yielded 36.45 mg/g and 3.13 mg/g. Analysis of the factorial identified the optimal conditions with an extraction time of 270 min and a pressure of 200 bar, the temperature had no effect within the tested ranges. The predicted TFC was 203.11 mg/g under optimal conditions and experimentally 230.48 mg/g. The antioxidant activity with 2,2-Diphenyl-1-picrylhydrazyl method of the extracts under optimum conditions was 49.21% while the CSE extract was 37.05%. The TLC analysis of the supercritical fluid extracts from the optimum conditions showed 5 majors flavonoids.
20.	Person, Alixander, et al. (författare) Novel Approach to Controlled Surface Modification in Textile Via Magnetic Cross- Linked Enzyme Aggregates (Clea) 2015 Ingår i: Tekstil ve Muhendis. - : UCTEA Chamber of Textile Engineers. - 2147-0510 .- 1300-7599. ; 22:97, s. 44-49 Tidskriftsartikel (refereegranskat)abstract In this proceeding, architecting novel magnetic Cross-Linked-Enzyme-Aggregates (CLEA)nanoparticles to effectively and controllably modify textile surfaces without damaging the fabric itself, will bediscussed. The efficiency is due to exponentially increased surface concentration of biocatalyst and to thechoice of aggregate size which controls the penetration depth and hence preventing the fabric disruption. Inaddition, due to the presence of magnetic nanoparticles, the continuous recovery and reusability makes thisdesign more economic and ecologic compared to the conventional chemical processes. The careful fabricationand functionalization of such a design with preliminary results will be presented in this contribution.
21.	Perzon, Alixander, et al. (författare) Cellulase cross-linked enzyme aggregates (CLEA) activities can be modulated and enhanced by precipitant selection 2017 Ingår i: Journal of Chemical Technology and Biotechnology. - : Wiley. - 0268-2575. ; 92:7, s. 1645-1649 Tidskriftsartikel (refereegranskat)abstract BACKGROUND: Crosslinked enzyme aggregates (CLEA) technology is a rapid and versatile method to produce immobilized enzymes via precipitation and cross-linking. A direct relationship between CLEA final activity and process parameters is however yet to be clarified. To address the issue, we have used a factorial design to test the formation and optimization of CLEA made from technical grade cellulase (EC 3.2.1.4). Three types of precipitant (ammonium sulfate, polyethylene glycol, tert-butyl alcohol) were used at varied levels of cross-linker concentration, cross-linking time, and temperature. RESULTS: It was found that the CLEAs produced with tert-butyl alcohol were inactive, whereas the polyethylene glycol- and ammonium sulfate-CLEA, recovered 29 and 17% of the free enzyme activity, respectively. Testing for re-usability, we observed that the polyethylene glycol-CLEA maintained 40% of the initial activity after four cycles, in contrast the ammonium sulfate-CLEA only maintained 10% of its activity after one cycle. CONCLUSION: Our study highlights the importance of evaluating the effect of the precipitant on final CLEA activity rather than re-solubilized enzyme activity. It was demonstrated that polyethylene glycol, despite not being able to precipitate the enzymes as readily as ammonium sulfate, resulted in better performing CLEA.
22.	Sanchez-Fernandez, Adrian, et al. (författare) Hydration in Deep Eutectic Solvents Induces Non-monotonic Changes in the Conformation and Stability of Proteins 2022 Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 144:51, s. 23657-23667 Tidskriftsartikel (refereegranskat)abstract The preservation of labile biomolecules presents a major challenge in chemistry, and deep eutectic solvents (DESs) have emerged as suitable environments for this purpose. However, how the hydration of DESs impacts the behavior of proteins is often neglected. Here, we demonstrate that the amino acid environment and secondary structure of two proteins (bovine serum albumin and lysozyme) and an antibody (immunoglobulin G) in 1:2 choline chloride:glycerol and 1:2 choline chloride:urea follow a re-entrant behavior with solvent hydration. A dome-shaped transition is observed with a folded or partially folded structure at very low (<10 wt % H2O) and high (>40 wt % H2O) DES hydration, while protein unfolding increases between those regimes. Hydration also affects protein conformation and stability, as demonstrated for bovine serum albumin in hydrated 1:2 choline chloride:glycerol. In the neat DES, bovine serum albumin remains partially folded and unexpectedly undergoes unfolding and oligomerization at low water content. At intermediate hydration, the protein begins to refold and gradually retrieves the native monomer-dimer equilibrium. However, ca. 36 wt % H2O is required to recover the native folding fully. The half-denaturation temperature of the protein increases with decreasing hydration, but even the dilute DESs significantly enhance the thermal stability of bovine serum albumin. Also, protein unfolding can be reversed by rehydrating the sample to the high hydration regime, also recovering protein function. This correlation provides a new perspective to understanding protein behavior in hydrated DESs, where quantifying the DES hydration becomes imperative to identifying the folding and stability of proteins.
23.	Singh, Manish, et al. (författare) Conductive and enzyme-like silk fibers for soft sensing application 2020 Ingår i: Biosensors and Bioelectronics. - : Elsevier BV. - 0956-5663. ; 150, s. 1-7 Tidskriftsartikel (refereegranskat)abstract A combination of supercritical carbon dioxide (scCO2) impregnation of pyrrole and sonochemical transformation of permanganate (KMnO4) was used to impart conductive and catalytic properties to silk fibers. The results indicated that the conductivity (from polypyrrole –PPy) and catalytic activities (from manganese dioxide –MnO2) were independent and complementary within the processing parameters used. The enhanced conductivity was attributed to scCO2 preferentially distributing the pyrrole monomers along with the silk internal fibrillar structure and hence, yielding a more linear PPy. The oxidative properties of the PPy-MnO2-silk hybrid showed an enzyme-like behavior for the degradation of hydrogen peroxide (H2O2) with a Km of about 13 mM and specific activity of 1470 ± 75 μmol/min/g. Finally, we demonstrated that the PPy-MnO2-silk hybrid could be used as soft working electrodes for the simultaneous degradation and detection of H2O2.
24.	Singh, Manish, et al. (författare) Manganese oxide functionalized silk fibers for enzyme mimics application 2020 Ingår i: Reactive and Functional Polymers. - : Elsevier BV. - 1381-5148. Tidskriftsartikel (refereegranskat)abstract The inorganic metal or metal-oxide nanoparticles (NPs) that mimic enzymes are of great interest due to improved physical and chemical properties compared with native enzymes. Here, we report that manganese dioxide (MnO2)-Silk exhibit catalase, oxidase, and peroxidase-like activities. The MnO2-Silk hybrid fibers effectively decomposed hydrogen peroxide (H2O2) and oxidized the typical horseradish peroxidase substrates, such as o-phenylenediamine (OPD), and 3,3′,5,5′- tetramethylbenzidine (TMB) in the presence or absence of H2O2. The oxidative properties of MnO2-Silk fiber hybrid showed an enzyme-like behavior for the catalase-like activity, oxidase-like activity, and peroxidase-like activity. The operational stability of the MnO2-Silk fiber hybrid over ten cycles showed a constant residual activity of about 25–30% after 2–3 cycles indicating that MnO2-Silk fiber hybrid could be used as a satisfactory oxidoreductase enzyme mimics. Potentiometric titration was used to determine the surface charges of the MnO2-Silk catalyst. Together, we identified the reactive species as Mn1−x4+Mnx3+O2−x(OH)x with a pK of approximately 5.2. Our results have implications on the understanding of the catalytic origin and interaction of metal oxides NP with various biomaterials.
25.	Singh, Manish, et al. (författare) Rapid fabrication and optimization of silk fibers supported and stabilized MnO2 catalysts 2017 Ingår i: Fibers and Polymers. - : Springer Science and Business Media LLC. - 1229-9197 .- 1875-0052. ; 18:9, s. 1660-1670 Tidskriftsartikel (refereegranskat)abstract We report on the in situ synthesis and stabilization of manganese dioxide (MnO2) onto four different silk yarns (mulberry, tasar, muga and eri silks). A new ultrasound-assisted procedure was used to reduce permanganate (MnO4 −) and yielded MnO2 nanoparticles (NPs) on/in the different silk fibers. Using a factorial design we assessed the influence of the silk type, manganese precursor concentration, sonication time, and temperature. The results indicated no measurable effect of the process parameters on the silk structures, but significant correlation with the rate of degradation of methylene blue (MB) and the fraction of permanganate consumed. Further optimization of the factorial model identified the optimal process conditions for each silks: mulberry (150 min sonication, 20 mM permanganate), eri (360 min, 10 mM), tasar (150 min, 10 mM) and Muga (20 min, 10 mM). The operational stability (successive catalysis) of the optimum hybrids showed good performance over 5 cycles and most importantly reduced direct dye absorption relatively to dye oxidation. Overall, we found that all silks could template the formation and stabilization of different MnO2 polymorphs and yielded catalytic instead of stoichiometric hybrid fibers.
26.	Singh, Manish, et al. (författare) Sonication enhances the stability of MnO2 nanoparticles on silk film template for enzyme mimic application 2020 Ingår i: Ultrasonics Sonochemistry. - : Elsevier BV. - 1350-4177. ; 64 Tidskriftsartikel (refereegranskat)abstract We have developed an in-situ method using sonication (3 mm probe sonicator, 30 W, 20 kHz) and auto-reduction (control) to study the mechanism of the formation of manganese dioxide (MnO2) on a solid template (silk film), and its resulting enzymatic activity on tetramethylbenzidine (TMB) substrate. The fabrication of the silk film was first optimized for stability (no degradation) and optical transparency. A factorial approach was used to assess the effect of sonication time and the initial concentration of potassium permanganate (KMnO4). The result indicated a significant correlation with a fraction of KMnO4 consumed and MnO2 formation. Further, we found that the optimal process conditions to obtain a stable silk film with highly catalytic MnO2 nanoparticles (NPs) was 30 min of sonication in the presence of 0.5 mM of KMnO4 at a temperature of 20–24 °C. Under the optimal condition, we monitored in-situ the formation of MnO2 on the silk film, and after thorough rinsing, the in-situ catalysis of 0.8 mM of TMB substrate. For control, we used the auto-reduction of KMnO4 onto the silk film after about 16 h. The result from single-wavelength analysis confirmed the different kinetics rates for the formation of MnO2 via sonication and auto-reduction. The result from the multivariate component analysis indicated a three components route for sonication and auto-reduction to form MnO2-Silk. Overall, we found that the smaller size, more mono-dispersed, and deeper buried MnO2 NPs in silk film prepared by sonication, conferred a higher catalytic activity and stability to the hybrid material.
27.	Singh, Manish, et al. (författare) Supercritical carbon dioxide impregnation of gold nanoparticles demonstrates a new route for the fabrication of hybrid silk materials 2022 Ingår i: Insects. - : MDPI AG. - 2075-4450. ; 13:1 Tidskriftsartikel (refereegranskat)abstract How many nanoparticles can we load in a fiber? How much will leak? Underlying is the relatively new question of the “space available” in fibers for nanoparticle loading. Here, using supercritical carbon dioxide (scCO2 ) as a carrier fluid, we explored the impregnation in four Indian silks (Mulberry, Eri, Muga, and Tasar) with five standard sizes of gold nanoparticles (5, 20, 50, 100 and 150 nm in diameter). All silks could be permanently impregnated with nanoparticles up to 150 nm in size under scCO2 impregnation. Accompanying structural changes indicated that the amorphous silk domains reorganized to accommodate the gold NPs. The mechanism was studied in detail in degummed Mulberry silk fibers (i.e., without the sericin coating) with the 5 nm nanoparticle. The combined effects of concentration, time of impregnation, scCO2 pressure, and temperature showed that only a narrow set of conditions allowed for permanent impregnation without deterioration of the properties of the silk fibers.
28.	Toytziaridis, Andreas, et al. (författare) Fabrication and optimization of stable, optically transparent, and reusable ph-responsive silk membranes 2016 Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 17:11 Tidskriftsartikel (refereegranskat)abstract The fabrication of silk-based membranes that are stable, optically transparent and reusable is yet to be achieved. To address this bottleneck we have developed a method to produce transparent chromogenic silk patches that are optically responsive to pH. The patches were produced by blending regenerated silk fibroin (RSF), Laponite RD (nano clay) and the organic dyes neutral red and Thionine acetate. The Laponite RD played a central role in the patch mechanical integrity and prevention of dye leaching. The process was optimized using a factorial design to maximize the patch response to pH by UV absorbance and fluorescence emission. New patches of the optimized protocol, made from solutions containing 125 µM neutral red or 250 µM of Thionine and 15 mg/mL silk, were further tested for operational stability over several cycles of pH altering. Stability, performance, and reusability were achieved over the tested cycles. The approach could be extended to other reporting molecules or enzymes able to bind to Laponite.
29.	Yang, Yuhong, et al. (författare) Behavior of silk protein at the air-water interface 2012 Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 8:37, s. 9705-9712 Tidskriftsartikel (refereegranskat)abstract The inability to link silk biomacromolecules' activity to their self-assembly and further fiber formation has limited a true implementation of a silk biotechnology. In this paper, we describe the application of video-enhanced drop shape analysis and interfacial shear rheological measurement to characterize the dynamic surface activity and interfacial interactions, as well as molecular structure within the interfacial layer of silk proteins. Quantitative analysis of the dynamic surface tension highlights two major mechanisms: (i) a mixed model at low concentration, and, (ii) a diffusion limited model at higher concentration. Once regenerated silk fibroin (RSF) is adsorbed at the air-water interface, interfacial gel-like structures are formed. The interfacial elastic modulus (G') of the adsorbed membranes exhibited a non-monotonic concentration dependence with a local maximum value at 1.0 x 10(-5) g mL(-1), indicating a different surface structure formed in RSF solution. The viscoelastic behavior varies with RSF concentration corresponding to three states of a soft glassy system, i. e. below, above and near the glass transition. A structural model for RSF adsorbed layers at the air-water interface at different bulk concentrations is suggested.
30.	Öster, Carl, et al. (författare) Characterization and assembly of a GFP-tagged cylindriform silk into hexameric complexes. 2014 Ingår i: Biopolymers. - : Wiley. - 0006-3525. ; 101:4, s. 378-390 Tidskriftsartikel (refereegranskat)abstract Spider silk has been studied extensively for its attractive mechanical properties and potential applications in medicine and industry. The production of spider silk, however, has been lagging behind for lack of suitable systems. Our approach focuses on solving the production of spider silk by designing, expressing, purifying and characterizing the silk from cylindriform glands. We show that the cylindriform silk protein, in contrast to the commonly used dragline silk protein, is fully folded and stable in solution. With the help of GFP as a fusion tag we enhanced the expression of the silk protein in Escherichia coli and could optimize the downstream processing. Secondary structures analysis by circular dichroism and FTIR shows that the GFP-Silk fusion protein is predominantly α-helical, and that pH can trigger a α- to β-transition resulting in aggregation. Structural analysis by small angle x-ray scattering suggests that the GFP-Silk exists in the form of a hexamer in solution.

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