Size Exclusion Chromatography Analysis Research Articles

Formulation of Multifunctional Materials Based on the Reaction of Glyoxalated Lignins and a Nanoclay/Nanosilicate.

Two organosolv lignins from different origins, namely, almond shells and maritime pine, were modified by using a nanoclay and nanosilicate. Prior to modification, they were activated via glyoxalation to enhance the reactivity of the lignins and thus ease the introduction of the nanoparticles into their structure. The lignins were characterized by several techniques (Fourier transformed infrared, high-performance size exclusion chromatography, 1H NMR, X-ray diffraction, and thermogravimetric analysis) before and after modification to elucidate the main chemical and structural changes. The reaction with glyoxal proved to increase the amount of hydroxyl groups and methylene bridges. This tendency was more pronounced, as the percentage of glyoxal was incremented. On the other side, the addition of the nanoclay and nanosilicate particles improved the thermal stability of the lignins compared to that of the original unmodified ones. This trend was more evident for the lignin derived from maritime pine, which displayed better results regarding the thermal stability, indicating a more effective combination of the nanoparticles in the lignin structure during the modification process.

Expression, purification, and efficient refolding of the extracellular domain of Escherichia coli-expressed signaling receptor herpesvirus entry mediator

Herpesvirus entry mediator (HVEM), a member of the TNF-receptor superfamily, plays an important role in the regulation of the immune system. It forms a complex with ligands and can either activate or inhibit the response of the immune system. Furthermore, HVEM can exhibit pro-inflammatory or anti-inflammatory effects in many human diseases. Therefore, understanding the mechanism underlying the interaction of HVEM with other receptors is extremely important to design small therapeutic molecules that can stimulate the response of the immune system.In this study, we attempted to develop the most efficient method for the expression and purification of the extracellular domain of HVEM using Escherichia coli. The soluble fraction constituted only a small portion of the E. coli-expressed protein, whereas majority of the protein was found to be accumulated in the insoluble fraction. Three different protein refolding methods were analyzed: dialysis, dilution, and using chromatographic column. The oligomeric state of the protein was determined by characterizing the obtained fractions using analytical size exclusion chromatography. All the obtained fractions were tested for their ability to form a complex with B- and T-lymphocyte attenuator using enzyme-linked immunosorbent assay.The results of this study provide crucial information regarding the production of HVEM protein in a robust, well-established, and convenient heterologous expression system using E. coli as a host. In addition, it allows for the selection of the most effective method for appropriate refolding of HVEM protein, which gets accumulated in the insoluble fraction.

Fusion with an albumin-binding domain improves pharmacokinetics of an αvβ3-integrin binding fibronectin scaffold protein.

Fusion with an albumin-binding domain (ABD) of streptococcal protein G represents a popular approach for half-life extension of small protein therapeutics in the organism. To increase the circulation time of engineered αvβ3-integrin-binding protein (JCL) based on the 10th human fibronectin type III domain (10 Fn3), we have constructed several fusions with ABD with different orientations of the partner proteins and linker length. The recombinant proteins were expressed in Escherichia coli cells and purified by nickel-affinity chromatography. All fusion proteins bound human serum albumin (HSA) in ELISA assay; however, fusions with longer linkers demonstrated better performance. Interaction of ABD-L15 -JCL and JCL-L14 -ABD with HSA was confirmed by analytical size exclusion chromatography and pull-down assays. Surprisingly, the thermal stability of ABD-L15 -JCL was dramatically decreased in comparison with JCL and JCL-L14 -ABD proteins. Pharmacokinetic studies revealed that JCL-L14 -ABD circulated in murine blood about 10 times longer than ABD-L15 -JCL and 960 times longer than JCL. Biodistribution studies of JCL-L14 -ABD in mice revealed its increased level in blood and a decreased accumulation in liver and kidneys in comparison with JCL. Obtained results demonstrate the utility of the fusion with ABD for half-life extension of the binding proteins based on 10 Fn3.

Abstract 4483: Novel small-molecule antagonists of the PD-1/PD-L1 axis that mediate cell surface PD-L1 dimerization and internalization

Abstract Blocking the PD-(L)1 immune checkpoint axis with therapeutic antibodies against either the receptor or the ligand has proven to be an effective treatment modality for multiple cancer histologies. We describe the identification and characterization of novel small molecule antagonists of the PD-(L)1 axis that function by inducing dimerization and subsequent internalization of the PD-L1 protein, effectively depleting the ligand from the cell membrane and preventing PD-1 activation on T cells. Compound-dependent PD-L1 dimerization was characterized using several biophysical techniques including fluorescence resonance energy transfer (FRET) measurements, size exclusion chromatography and thermal shift analysis. Experimental evidence demonstrates compound-dependent dimer conformation with slow dissociation kinetics and significantly enhanced thermal stability. Many of the PD-L1-directed small molecules blocked binding of soluble PD-1 to either native PD-L1 expressed on cancer cell lines or PD-L1 expressed in CHO cells with low nanomolar potency. However, only a subset of the small molecules caused loss of cell surface PD-L1 in a time- and concentration-dependent manner. Importantly, there was a strict correlation between the promotion of PD-L1 internalization secondary to dimerization and the induction of an NFAT response element-luciferase reporter gene. Strikingly, only those small molecules that could produce a specific dimeric PD-L1 conformation as measured using FRET were associated with functional activity in cells, suggesting that PD-L1 dimerization was necessary but not sufficient for internalization and cellular activity. A cell-active tool compound (cell binding IC50 <5 nM, internalization EC50 <10 nM) was fluorescently labeled to enable direct visualization of intracellular trafficking. Confocal microscopy with this PD-L1 antagonist showed time-dependent increases in intracellular fluorescence in PD-L1 expressing, but not PD-L1 deleted, cells. The internalized antagonist showed punctate staining coincident with markers of the early endosome, and independent studies confirmed that the internalized PD-L1 also trafficked to the early endosome. By disrupting the suppressive activity of PD-L1 on PD-1, these inhibitors result in functional activation of T cells in ex vivo cellular assays in a manner equivalent to antibodies directed against either PD-1 or PD-L1. In summary, we have identified a series of potent, small molecule PD-L1 antagonists that induce dimerization of the protein; inhibitors that trigger an appropriate dimeric conformation can also induce PD-L1 internalization thereby alleviating PD-L1-induced suppression of T cell activation. Citation Format: Phillip C.C. Liu, Richard Wynn, Liangxing Wu, Alla Volgina, Nina Zolotarjova, Luping Lin, Pramod Thekkat, Alex Margulis, Ronald Klabe, Wenqing Yao, Kaijiong Xiao, Jingwei Li, Xin He, Mark Rupar, Hong Chang, Paul Waeltz, Yanlong Li, Peggy Scherle, Reid Huber, Gregory Hollis. Novel small-molecule antagonists of the PD-1/PD-L1 axis that mediate cell surface PD-L1 dimerization and internalization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4483.

Insights into the full-length SRPK2 structure and its hydrodynamic behavior

The serine/arginine-rich protein kinase 2 (SRPK2) has been reported as upregulated in several cancer types, with roles in hallmarks such as cell migration, growth, and apoptosis. These findings have indicated that SRPK2 is a promising emerging target in drug discovery initiatives. Although high-resolution models are available for SRPK2 (PDB 2X7G), they have been obtained with a heavily truncated recombinant protein version (~50% of the primary structure), due to the presence of long intrinsically unstructured regions. In the present work, we sought to characterize the structure of a full-length recombinant version of SRPK2 in solution. Low-resolution Small-Angle X-ray Scattering data were obtained for both versions of SRPK2. The truncated ΔNΔS-SRPK2 presented a propensity to dimerize at higher concentrations whereas the full-length SRPK2 was mainly found as dimers. The hydrodynamic behavior of the full-length SRPK2 was further investigated by analytical size exclusion chromatography and sedimentation velocity analytical ultracentrifugation experiments. SRPK2 behaved as a monomer-dimer equilibrium and both forms have an elongated shape in solution, pointing to a stretched-to-closed tendency among the conformational plasticity observed. Taken together, these findings allowed us to define unique structural features of the SRPK2 within SRPK family, characterized by its flexible regions outside the bipartite kinase domain.

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS).

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

Molecular weight determination of heparosan- and chondroitin-like capsular polysaccharides: figuring out differences between wild -type and engineered Escherichia coli strains

Heparin and chondroitin sulfate are used as anti-thrombic and anti-osteoarthritis drugs, respectively, but their pharmacological actions depend on their structural characteristics such as their sulfation grade and their molecular weight. In the last years, new fermentation-based biotechnological approaches have tried to obtain heparin and chondroitin sulfate starting from the heparosan and chondroitin-like capsular polysaccharides produced by Escherichia coli K5 and K4. The study of the microbial capsular polysaccharide molecular weight is critical to obtain nature-like or structural tailor cut glycosaminoglycan homologues. However, so far, it has been scarcely investigated. In this paper, for the first time, a new protocol was set up to determine the molecular weights of the capsular polysaccharides of three wild-type and three engineered E. coli K5 and K4 strains. The protocol includes a small-scale downstream train to purify the intact polysaccharides, directly from the fermentation broth supernatants, by using ultrafiltration membranes and anion exchange chromatography, and it couples size exclusion chromatography analyses with triple detector array. In the purification high recovery (> 85.0%) and the removal of the main contaminant, the lipopolysaccharide, were obtained. The averaged molecular weights of the wild-type capsular polysaccharides ranged from 51.3 to 90.9kDa, while the engineered strains produced polysaccharides with higher molecular weights, ranging from 68.4 to 130.6kDa, but with similar polydispersity values between 1.1 and 1.5.

Utilization of Complex Pectic Polysaccharides from New Zealand Plants (Tetragonia tetragonioides and Corynocarpus laevigatus) by Gut Bacteroides Species.

Pectic polysaccharides from New Zealand (NZ) spinach (Tetragonia tetragonioides) and karaka berries (Corynocarpus laevigatus) were extracted and analyzed. NZ spinach polysaccharides comprised mostly homogalacturonan (64.4%) and rhamnogalacturonan I (5.8%), with side chains of arabinan (8.1%), galactan (2.2%), and type II arabinogalactan (7.1%); karaka berry polysaccharides comprised homogalacturonan (21.8%) and rhamnogalacturonan I (10.0%), with greater proportions of side chains (arabinan, 15.6%; galactan, 23.8%; and type II arabinogalactan, 19.3%). Screening of gut commensal Bacteroides showed that six were able to grow on the NZ spinach extract, while five were able to grow on the karaka berry extract. Analysis of the polysaccharides remaining after fermentation, by size-exclusion chromatography and constituent sugar analysis, showed that the Bacteroides species that grew on these two substrates showed preferences for the different pectic polysaccharide types. Our data suggest that, to completely degrade and utilize the complex pectin structures found in plants, members of Bacteroides and other bowel bacteria work as metabolic consortia.

Functional dextran amino acid ester particles derived from N-protected S-trityl-L-cysteine

This work describes the derivatization of dextran using N-(tert-butyloxycarbonyl)-S-(trityl)-L-cysteine in the presence of N,N′-carbonyldiimidazole (CDI) as a coupling agent. Homogeneous reactions in dimethyl sulfoxide allowed for an efficient coupling of the amino acid derivative to the polymer backbone. Derivatization was confirmed by infrared and 13C NMR spectroscopy, size exclusion chromatography and elemental analysis. The presence of hydrophobic protecting groups resulted in a product that can be shaped into water-insoluble particles stable in an aqueous environment and non-toxic for lung epithelial cells. It is suggested that materials composed of ester bonds between amino acids and polysaccharides are useful for targeted drug delivery, bio-imaging or surface functionalization.

Cyclic Hydrazide‐Functionalized Poly(ethylene oxide) Frameworks for the Synthesis of pH‐Cleavable Drug‐Carriers and Their Applications for the Stabilization of Gold Nanoparticles

AbstractIn this paper, two different types of poly(ethylene oxide) (PEO) frameworks with different functional groups such as the thiol group and cyclic hydrazide in the α,ω‐positions bearing “smartness” are introduced. These heterobifunctional PEOs are synthesized via different functionalization approaches using t‐butoxy PEO. Heterobifunctional PEOs, both α‐luminol‐ω‐thiol PEO and α‐thiol‐ω‐cyclic hydrazide PEO, are prepared by chain‐end functionalization of the reactive t‐butoxy PEOs. The chain‐end luminol as a cyclic hydrazide is found to be effective to yield pH‐responsive prodrugs from the reaction with doxorubicin (Dox) yielding corresponding Dox‐tethered PEO. The active t‐butoxy PEO‐initiated block copolymerization of N‐phenylmaleimide (N‐PMI) in acetone yields a block copolymer controlled in the 3‐ to 5‐units range of the N‐PMI group. The deprotection of the t‐butoxy group, followed by tosylation, thioacetylaton, and the Gabriel process, provides corresponding α‐thiol‐ω‐cyclic hydrazide PEO. The functionality yields are almost quantitative (over 98 mol%). Polymeric prodrugs such as Dox‐tethered PEO and folate‐conjugated PEO are successfully employed for the stabilization of gold (Au) nanoparticles. The resulting products are characterized by a combination of proton nuclear magnetic resonance (1H NMR) spectroscopic, ultraviolet (UV)–visible spectroscopic, Fourier transform infrared (FT‐IR), transmission electron microscopic (TEM), and size‐exclusion chromatographic (SEC) analysis.

Changes in physicochemical and structural properties of tapioca starch after high speed jet degradation

Tapioca starch (TS) suspensions (10%, w/v) were subjected to high speed jet (HSJ) processing at 80, 120, 160, 200 and 240 MPa for one pass, and the effects of HSJ treatment on solubility, rheological, pasting, morphological and molecular properties were further investigated. Polarized light microscopy observation revealed TS was partly gelatinized, and the number and clarity of“Maltese cross” decreased with the increase of HSJ pressure. The solubility of TS was increased significantly from 1.59% (0 MPa) to 12.11% (240 MPa). Dynamic oscillation analysis showed that TS gels displayed less elastic and more liquid like after HJS treatment, and all samples displayed pseudo-plastic properties during flow properties analysis. Rapid Visco Analyser measurement indicated that treated samples showed significantly decreased in peak viscosity and exhibited a lower tendency towards retrogradation on cooling process compared to untreated TS. Size-exclusion chromatography analysis suggested that the degradation of TS happened during HSJ treatment, and the degradation was more prominent beyond 160 MPa. The results showed that the significant changed in physicochemical and structural properties of treated TS were related to its degradation. These observations would provide an alternative and environmentally approach for starch modification.

Oligomeric stability of Glossoscolex paulistus hemoglobin as a function of the storage time.

Glossoscolex paulistus hemoglobin structure is composed of 144 globin chains and 36 polypeptide chains lacking the heme group, with a total molecular mass of 3600 kDa. The current study focuses on the oxy-HbGp oligomeric stability, as a function of the storage time, at pH 7.0, using dynamic light scattering, analytical ultracentrifugation (AUC), optical absorption and size exclusion chromatography (SEC). HbGp stored in Tris-HCl buffer, pH 7.0, at 4 °C, for two years remains in the native form, while 4-6 years HbGp stocks present typical hemichrome species absorption spectra. AUC and SEC analyses show that the contribution of HbGp-subunits, such as, dodecamer (abcd)3, tetramer abcd, trimer abc and monomer d, increases with the protein aging due to the lower stability of the HbGp with the time. The dissociation and the oxidation of the iron noted for the older protein solutions indicate that HbGp storage for periods of time longer than two years changes its ability to carry oxygen. Despite the reduction of HbGp stability and oxygen carrying capacity with aging, the protein stability is still larger as compared to mammalian hemoglobins. Thus, the extracellular hemoglobins are quite stable and resistant to the auto-oxidation process, making them of interest for biotechnological applications.

Assessment of XXT1 and XXT5 Modes of Substrate Binding and Structure‐Solubility Relationship Using Site‐Directed Mutagenesis

Plant cell wall polysaccharides are the largest source of biopolymers on Earth and have numerous industrial applications such as food, fiber, biofuels and biomaterials. However, development is impeded because of the dearth of structural information for plant glycosyltransferases, the key enzymes responsible for polysaccharide biosynthesis. A set of specific transmembrane proteins synthesizes the most abundant hemicellulosic polysaccharide, xyloglucan. Two Xyloglucan Xylosyltransferases 1 and 5 (XXT1 and XXT5, respectively) add a xylose moiety to the glucan backbone, as the initial step in xyloglucan branching. The structure of XXT1 was solved using X‐ray crystallography and key information about residues involved in catalysis and protein solubility was obtained through site‐directed mutagenesis, size exclusion chromatography (SEC), and activity assays using a newly developed PKLD enzyme assay.In order to reveal modes of substrate binding and ascertain the conclusions drawn from the crystal structure of XXT1, site directed mutagenesis was performed to assess the critical amino acids involved in glycosyltransferase activity. The mutation of the residues involved in coordination of Mn2+ such as Asp229Ala, Asp227Ala/Asp229Ala, and His337Ala, significantly reduced enzyme activity. Mutations Lys382Ala, Asp317Ala, Asp318Ala, and Gln319Ala reduced activity most due to their importance in hydrogen bonding. Whereas, Ser228Ala and Asn268Ala mutants did not show changes in activity.The next objective of our study was to investigate the suitability of XXT5 for crystallization. The apparent lower solubility of XXT5 compared with XXT1 prevents the former protein to be crystallized. First, a homology model of XXT5 has been generated from the crystal structure of XXT1 and differences in surface amino acids were revealed. By substituting the revealed residues in XXT5 to those present at the same positions in XXT1, we anticipate to disrupt some protein‐protein interactions, thus reducing aggregation and increasing protein solubility. Vectors harboring the mutated gene were transformed into BL21 E. coli expression cells and, after expression and purification, mutant protein yield will be quantified using SDS‐PAGE. Those mutants that had higher expression in comparison with wild type XXT5 are currently being assessed using SEC analysis. The mutants showing higher solubility will be assayed for enzymatic activity to confirm the mutation did not affect protein activity. If the point mutation has shown sufficient expression, suitable solubility, and high activity, the screen for protein crystallization will be initiated.Solving the crystal structure of XXT1 and characterizing mutants allowed us to propose the model of xylosylation patterns in native xyloglucans. Structural information about the xyloglucan‐synthesizing enzymes is critical for our understanding of polysaccharide biosynthesis in plants and will have high impact on the development of plant biomass with improved properties for agriculture and industrial applications.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An integrated strategy for the effective production of bristle protein hydrolysate by the keratinolytic filamentous bacterium Amycolatopsis keratiniphila D2

In a conventional microorganism-mediated biological process for degradation of keratinous waste material the production of keratin-specific proteases (i.e., keratinases) and the hydrolysis of keratin-rich residual biomass both take place during the same stage of the bioprocess and, as a consequence, occur simultaneously under suboptimal conditions. In the present study the keratinolytic actinomycete Amycolatopsis keratiniphila D2 was successfully employed to biodegrade thermally pretreated porcine bristles at high solids loading (16% w/v) via a novel cultivation methodology. Indeed, the two-stage submerged fermentation process developed in this work enabled to efficiently recover, in a single unit operation, about 73% of the protein material contained in the keratinous biowaste structure, resulting in an overall accumulation of 89.3 g·L−1 protein-rich hydrolysate and a productivity of 427 mg crude soluble proteins per litre per hour. The obtained protein hydrolysate powder displayed a 2.2-fold increase in its in vitro pepsin digestibility (95%) with respect to the non-hydrolysed pretreated substrate (43%). In addition, the chromatogram obtained by size-exclusion chromatography analysis of the final product indicated that, among the identified fractions, those consisting of small peptides and free amino acids were the most abundantly present inside the analysed sample. Given these facts it is possible to conclude that the soluble proteins, peptides and free amino acids recovered through the newly designed two-stage bioextraction process could represent a viable alternative source of protein in animal feed formulation.

The synthesis, characterization and effect of molar mass distribution on solid-state degradation kinetics of oligo(orcinol)

In this study, the oxidative polymerization of orcinol monohydrate using different oxidants such as NaOCl, H2O2, and air was investigated. Polymerization studies were carried out between 50 and 90 °C. The optimum reaction conditions of the polymerization were also established. NaOCl was found to be the most active oxidant. The characterization of oligo(orcinol) was conducted by using FT-IR, 1H-NMR and 13C-NMR, TGA, size exclusion chromatography (SEC) and solubility techniques. At the optimum reaction conditions, the conversion to oligomer of orcinol was found to be 62% (for NaOCl oxidant), 42% (for H2O2 oxidant), and 21% (for air oxidant). According to the SEC analysis, the number-average molecular mass (Mn), mass-average molecular mass (Mw) and polydispersity index (PDI) values of oligo(orcinol) were determined to be 2260, 2540 g mol−1, and 1.12, using NaOCl, and 2170, 2470 g mol−1, and 1.14, using H2O2 and 1500, 1770 g mol−1, and 1.18, using air, respectively. In addition, the relationship between molar mass distributions and activation energies of thermal degradation processes of oligo(orcinol) was investigated by using TG analysis. For this purpose, the methods based on multiple heating rates such as Flynn–Wall–Ozawa [FWO], Tang, and Kissinger–Akahira–Sunose [KAS] were used. The activation energy related to the solid-state decomposition of oligo(orcinol) synthesized with NaOCl oxidant was calculated to be 79.02 kJ mol−1 by KAS method, 78.74 kJ mol−1 by Tang method and 81.78 kJ mol−1 by FWO method in the range of 0.05 < α < 0.95. The results obtained show that activation energy increased with an increase in molar mass.

Changes in Nanoscale Chain Assembly in Sweet Potato Starch Lamellae by Downregulation of Biosynthesis Enzymes.

Granule-bound starch synthase I (GBSSI) and starch-branching enzymes I and II (SBEI and SBEII) are crucial enzymes that biosynthesize starches with varied apparent amylose contents and amylopectin branching structure. With a sweet potato ( Ipomoea batatas [L.] Lam. cv. Xushu22), this work shows that downregulating GBSSI (for waxy starch) or SBE (for high-amylose starch) activity allowed the formation of new semicrystalline lamellae (named Type II) in sweet potato starch in addition to the widely reported Type I lamellae. Small-angle X-ray scattering (SAXS) results show that, compared with Type I lamellae, Type II lamellae displayed increased average thickness and thickness-distribution width, with thickened amorphous and crystalline components. The size-exclusion-chromatography (SEC) data revealed mainly two enzyme sets, (i) and (ii), synthesizing amylopectin chains. Reducing the GBSSI or SBE activity increased the amounts of amylopectin long chains (degree of polymerization (DP) ≥ 33). Combined SAXS and SEC analyses indicate that parts of these long chains from enzyme set (i) could be confined to Type II lamellae, followed by DP ≤ 32 short chains in Type I lamellae and the rest of the long chains from enzyme sets (i) and (ii) spanning more than a single lamella.

Polyamide-6 structuration induced by a chemical reaction with a polyether triamine in the molten state

Polyamide-6 was modified by reactive extrusion using a triamine as a structuring agent. To scrutinize the reactive medium after the chemical modification and to characterize the new structures obtained, the coupling of several physico-chemical analyses was necessary and applied to either model molecules or polyamide chain. By correlation of liquid NMR, matrix assisted laser desorption ionisation/time of flight mass spectrometry (MALDI-ToF MS), and size-exclusion chromatography (SEC) analyses, it was possible to observe both grafting and polyamide chain scissions that led to randomly branched and linear molecules. One of the most noticeable modifications regarding the properties was a decrease in the amorphous phase mobility with a very small amount of triamine (0.7 wt%). A comprehensive study by X-ray scattering techniques (small and wide angle X-ray scattering, SAXS and WAXS, respectively) allowed us to highlight the noticeable size evolution of the crystalline and amorphous parts at the lamellar scale. In conclusion, these multi-scale experiments demonstrate the possibility of modifying the amorphous phase mobility by controlling the nanostructure morphology of the material, particularly the crystalline lamellae thickness and the rigid amorphous fraction (RAF) content. Thus, an increase in the RAF content of around 6 % induced more restricted mobility in the amorphous phase as the glass transition temperature (Tg) shifted to higher temperature (≈ + 7 °C).

Bacterial conversion of depolymerized Kraft lignin

BackgroundLignin is a potential feedstock for microbial conversion into various chemicals. However, the microbial degradation rate of native or technical lignin is low, and chemical depolymerization is needed to obtain reasonable conversion rates. In the current study, nine bacterial strains belonging to the Pseudomonas and Rhodococcus genera were evaluated for their ability to grow on alkaline-treated softwood lignin as a sole carbon source.ResultsPseudomonas fluorescens DSM 50090 and Rhodococcus opacus DSM1069 showed the best growth of the tested species on plates with lignin. Further evaluation of P. fluorescens and R. opacus was made in liquid cultivations with depolymerized softwood Kraft lignin (DL) at a concentration of 1 g/L. Size-exclusion chromatography (SEC) showed that R. opacus consumed most of the available lower-molecular weight compounds (approximately 0.1–0.4 kDa) in the DL, but the weight distribution of larger fractions was almost unaffected. Importantly, the consumed compounds included guaiacol—one of the main monomers in the DL. SEC analysis of P. fluorescens culture broth, in contrast, did not show a large conversion of low-molecular weight compounds, and guaiacol remained unconsumed. However, a significant shift in molecular weight distribution towards lower average weights was seen after cultivation with P. fluorescens.ConclusionsRhodococcus opacus and P. fluorescens were identified as two potential microbial candidates for the conversion/consumption of base-catalyzed depolymerized lignin, acting on low- and high-molecular weight lignin fragments, respectively. These findings will be of relevance for designing bioconversion of softwood Kraft lignin.

In-solution behavior and protective potential of asparagine synthetase A from Trypanosoma cruzi

l-Asparagine synthetase (AS) acts in asparagine formation and can be classified into two families: AS-A or AS-B. AS-A is mainly found in prokaryotes and can synthetize asparagine from ammonia. Distinct from other eukaryotes, Trypanosoma cruzi produces an AS-A. AS-A from Trypanosoma cruzi (Tc-AS-A) differs from prokaryotic AS-A due to its ability to catalyze asparagine synthesis using both glutamine and ammonia as nitrogen sources. Regarding these peculiarities, this work uses several biophysical techniques to provide data concerning the Tc-AS-A in-solution behavior. Tc-AS-A was produced as a recombinant and purified by three chromatography steps. Circular dichroism, dynamic light scattering, and analytical size exclusion chromatography showed that Tc-AS-A has the same fold and quaternary arrangement of prokaryotic AS-A. Despite the tendency of protein to aggregate, stable dimers were obtained when solubilization occurred at pH ≤ 7.0. We also demonstrate the protective efficacy against T. cruzi infection in mice immunized with Tc-AS-A. Our results indicate that immunization with Tc-AS-A might confer partial protection to infective forms of T. cruzi in this particular model.

Functionalized Artificial Bidomain Proteins Based on an α-Solenoid Protein Repeat Scaffold: A New Class of Artificial Diels–Alderases

αRep is a family of entirely artificial repeat proteins. Within the previously described αRep library, some variants are homodimers displaying interdomain cavities. Taking advantage of these properties, one of these homodimers called αRep A3 was converted into entirely artificial single chain bidomain metalloenzymes. A nonmutated A3 domain was covalently linked with an A3′ domain bearing a unique cysteine on a chosen mutated position (F119C or Y26C). This single mutation ensured the covalent coupling of a 1:1 copper(II)/phenanthroline or copper(II)/terpyridine complex as a catalytic center within the interdomain cavity which was maintained large enough to accommodate two substrates of the Diels–Alder (D–A) reaction. This allowed us to obtain four new artificial Diels–Alderases that were fully characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, UV–vis spectroscopy, and size exclusion chromatography analyses and were then further used for the catalysis of the D–A r...