L-asparaginase (ASNase) is an amidohydrolase that can be used as a biopharmaceutical, as an agent for acrylamide reduction, and as an active molecule for L-asparagine detection. However, its free form displays some limitations, such as the enzyme's single use and low stability. Hence, immobilization is one of the most effective tools for enzyme recovery and reuse. Silica is a promising material due to its low-cost, biological compatibility, and tunable physicochemical characteristics if properly functionalized. Ionic liquids (ILs) are designer compounds that allow the tailoring of their physicochemical properties for a given task. If properly designed, bioconjugates combine the features of the selected ILs with those of the support used, enabling the simple recovery and reuse of the enzyme. In this work, silica-based supported ionic liquid-like phase (SSILLP) materials with quaternary ammoniums and chloride as the counterion were studied as novel supports for ASNase immobilization since it has been reported that ammonium ILs have beneficial effects on enzyme stability. SSILLP materials were characterized by elemental analysis and zeta potential. The immobilization process was studied and the pH effect, enzyme/support ratio, and contact time were optimized regarding the ASNase enzymatic activity. ASNase-SSILLP bioconjugates were characterized by ATR-FTIR. The bioconjugates displayed promising potential since [Si][N
Abstract The wide application of protein fibrils as functional materials has been restricted by the limited scalability of fibrillation methods, slow kinetics, and use of expensive purified proteins. Herein, inspired by the biological cooperativity of proteins in macro-molecularly crowded environments, these restrictions have been overcome. Using ionic liquid cholinium tosylate that acts as a fibrillation agent, instantaneous production of protein fibrils is shown directly from a real and low-cost matrix, i.e. egg white. The fibrillation of egg white proteome is confirmed by microscopy, whereas the fibrillation kinetics is monitored by fluorescence changes of the thioflavin T dye and secondary structural transitions. Spectroscopic and molecular docking studies are used to identify the proteins involved and to appraise the molecular-level mechanisms ruling the proteins structural changes upon fibrillation. The obtained fibrils have enhanced mechanical stiffness and cytocompatibility, demonstrating their potential to act as improved enzyme supports.
In the past years, large efforts have been placed in the development of novel separation techniques with improved resolution, simplicity, speed and easy to scale-up. Among these, ionic-liquid-based (IL-based) aqueous biphasic systems (ABS) have been broadly proposed for the separation of high-value compounds, allowing improved extraction performance and purification. More recently, significant efforts have been arranged on the synthesis and use of novel ILs with both an acceptable environmental footprint and enhanced biocompatibility. In this sense, this work aims to characterize ABS composed of cholinium carboxylate ILs ([Ch][CnO2], with n = 2 to 7), K3PO4 and water. The respective ternary phase diagrams, including binodal curves, tie-lines and tie-line lengths, were determined at (298 ± 1) K and at atmospheric pressure. The ability to form ABS (or of the IL to be salted-out) increases with the increase of the alkyl chain length of the IL anion, up to [Ch][C5O2]; nevertheless, for longer anion alkyl chain lengths ([Ch][C6O2] and [Ch][C7O2]) the ILs self-aggregation leads to a decrease of the ILs ability to form ABS. The liquid−liquid equilibrium data experimentally determined were modeled using the local composition activity coefficient model NRTL (Non-Random Two Liquid). Finally, the partition behavior of three alkaloids (nicotine, caffeine and theobromine), used here as hydrophobicity probes, was evaluated. In all studied systems, alkaloids preferentially migrate to the IL-rich phase, with partition coefficients (K) ranging between 2.23 and complete extraction, in a single-step. Furthermore, the set of ILs investigated allowed identifying an odd-even effect in the alkaloids partitioning derived from the IL anion alkyl chain length. These results support the salting-out effect exerted by K3PO4 and favorable dispersive interactions established between the IL-rich phase-forming components and the alkaloids.
Abstract Intracellular heme formation and trafficking are fundamental processes in living organisms. Three biogenesis pathways are used by bacteria and archaea to produce iron protoporphyrin IX (heme b ) that diverge after the formation of the common intermediate uroporphyrinogen III (uro’gen III). In this work, we identify and provide a detailed characterization of the enzymes involved in the transformation of uro’gen III into heme. We show that in this organism operates the protoporphyrin-dependent pathway (PPD pathway), in which the last reaction is the incorporation of ferrous iron into the porphyrin ring by the ferrochelatase enzyme. In general, following this final reaction, little is known about how the formed heme b reaches the target proteins. In particular, the chaperons that are thought to be required to traffic heme for incorporation into hemeproteins to avoid the cytotoxicity associated to free heme, remain largely unidentified. We identified in C. jejuni a chaperon-like protein, named CgdH2, that binds heme with a dissociation constant of 4.9 ± 1.0 µM, a binding that is impaired upon mutation of residues histidine 45 and 133. We show that C. jejuni CgdH2 establishes protein-protein interactions with ferrochelatase, which should enable for the observed transfer of heme from ferrochelatase to CgdH2. Phylogenetic analysis revealed that C. jejuni CgdH2 is evolutionarily distinct from the currently known chaperones. Therefore, CgdH2 is a novel chaperone and the first protein identified as an acceptor of the intracellularly formed heme, thus enlarging our understanding of bacterial heme homeostasis.
Nanomaterials have been extensively used in different applications due to their peculiar characteristics and nanoscale dimensions. Among nanoparticles, carbon-based nanomaterials are becoming highly attractive for biomedical applications such as diagnosis, tissue engineering, drug delivery, and biosensing. The conjugation of carbon-based nanomaterials with antibodies combines the properties of these materials with the specific and selective recognition ability of the antibodies to antigens. The present work proposes a process intensification approach for immunoglobulin G (IgG present in rabbit serum) attachment on multi-walled carbon nanotubes (MWCNTs) in a single step. The effect of several parameters, namely MWCNTs external diameter, rabbit serum concentration, MWCNTs functionalization and pH value, on the IgG attachment yield was evaluated. The dilution of rabbit serum decreased other protein attachment, namely rabbit serum albumin (RSA), while increasing the IgG yield to 100%. The interaction mechanisms between IgG and MWCNTs were evaluated at pH 5.0 to 8.0. The protonation of IgG amino acids indicates that N-term are the most reactive amino acids in the antibody structure. The identification of the N-term reactivity at pH 8.0 allows to indicate a possible orientation of the antibody over the MWCNTs surface, described as "end-on". Since the amount of RSA attached to MWNT decreased with the increase in serum dilution, the IgG orientation and amine activity was not affected. This orientation demonstrates that the IgG attachment over the surface of the MWCNTs could be an effective strategy to maintain the antigen recognition by the antibody, and to be used for biomedical applications.
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