Matheus M. Pereira

Enhanced extraction of proteins using cholinium-based ionic liquids as phase-forming components of aqueous biphasic systems

Aqueous biphasic systems (ABS) composed of ionic liquids (ILs) are promising platforms for the extraction and purification of proteins. In this work, a series of alternative and biocompatible ABS composed of cholinium-based ILs and polypropylene glycol were investigated. The respective ternary phase diagrams, tie-lines, tie-line lengths and critical points were determined at 25°C. The extraction performance of these systems for commercial bovine serum albumin (BSA) was then evaluated. The stability of BSA at the IL-rich phase was ascertained by size exclusion high-performance liquid chromatography and Fourier transform infrared spectroscopy. Appropriate ILs lead to the complete extraction of BSA for the IL-rich phase, in a single step, while maintaining the proteins native conformation. Furthermore, to evaluate the performance of these systems when applied to real matrices, the extraction of BSA from bovine serum was additionally carried out, revealing that the complete extraction of BSA was maintained and achieved in a single step. The remarkable extraction efficiencies obtained are far superior to those observed with typical polymer-based ABS. Therefore, the proposed ABS may be envisaged as a more effective and biocompatible approach for the separation and purification of other value-added proteins.

Enhanced extraction of bovine serum albumin with aqueous biphasic systems of phosphonium- and ammonium-based ionic liquids

Novel aqueous biphasic systems (ABS) composed of phosphonium- or ammonium-based ionic liquids (ILs), combined with a buffered aqueous solution of potassium citrate/citric acid (pH=7.0), were investigated for the extraction of proteins. For that purpose, the phase diagrams, tie-lines and tie-line lengths were determined at 25 °C, and the performance of these ABS for the extraction of bovine serum albumin (BSA) was then evaluated. The obtained results reveal that, with the exception of the more hydrophobic ILs, most of the systems investigated allow the complete extraction of BSA for the IL-rich phase in a single-step. These remarkable extraction efficiencies are far superior to those afforded by more conventional extraction systems previously reported. The composition of the biphasic systems, i.e., the amount of phase-forming components, was also investigated aiming at reducing the overall costs of the process without losing efficiency on the protein extraction. It is shown that the extraction efficiencies of BSA are maintained at 100% up to high protein concentrations (at least up to 10 g L(-1)). The recovery of the BSA from the IL-rich phase by dialysis is also shown in addition to the demonstration of the IL recyclability and reusability, at least for 3 times. In the sequential three-step extractions (BSA recovery/IL reusability), the extraction efficiencies of BSA for the IL-rich phase were maintained at 100%. For the improved ABS, the preservation of the protein native conformation was confirmed by Size Exclusion High-Performance Liquid Chromatography (used also as the quantification method) and by Fourier Transform Infra-Red spectroscopy. According to the results herein reported, ABS composed of phosphonium- or ammonium-based ILs and a biodegradable organic salt represent an alternative and remarkable platform for the extraction of BSA and may be extended to other proteins of interest.

Long-term protein packaging in cholinium-based ionic liquids: improved catalytic activity and enhanced stability of cytochrome c against multiple stresses

There is a considerable interest in the use of structurally stable and catalytically active enzymes, such as cytochrome C (Cyt C), in the pharmaceutical and fine chemical industries. However, harsh process conditions, such as temperature, pH, and presence of organic solvents, are the major barriers to the effective use of enzymes in biocatalysis. Herein, we demonstrate the suitability of bio-based ionic liquids (ILs) formed by the cholinium cation and dicarboxylate-based anions as potential media for enzymes, in which remarkable enhanced activity and improved stability of Cyt C against multiple stresses were obtained. Among the several bio-ILs studied, an exceptionally high catalytic activity (> 50-fold) of Cyt C was observed in aqueous solutions of cholinium glutarate ([Ch][Glu]; 1g/mL) as compared to the commonly used phosphate buffer solutions (pH 7.2), and > 25-fold as compared to aqueous solutions of cholinium dihydrogen phosphate ([Ch][Dhp]; 0.5g/mL) -the best known IL for long term stability of Cyt C. The catalytic activity of the enzyme in presence of bio-ILs was retained against several external stimulus, such as chemical denaturants (H2O2 and GuHCl), and temperatures up to 120 °C. The observed enzyme activity is in agreement with its structural stability, as confirmed by UV-Vis, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopies. Taking advantage of the multi-ionization states of di/tri-carboxylic acids, the pH was switched from acidic to basic by the addition of the corresponding carboxylic acid and choline hydroxide, respectively. The activity was found to be maximum at a 1:1 ratio of [Ch][carboxylate], with a pH in the range from 3 to 5.5. Moreover, it was found that the bio-ILs studied herein protect the enzyme against protease digestion and allow long-term storage (at least for 21 weeks) at room temperature. An attempt by molecular docking was also made to better understand the efficacy of the investigated bio-ILs towards the enhanced activity and long term stability of Cyt C. The results showed that dicarboxylates anions interact with the active sites amino acids of the enzyme through H-bonding and electrostatic interactions, which are responsible for the observed enhancement of the catalytic activity. Finally, it is demonstrated that Cyt C can be successfully recovered from the aqueous solution of bio-ILs and reused without compromising its yield, structural integrity and catalytic activity, thereby overcoming the major limitations in the use of IL-protein systems in biocatalysis.

Characterization of Biocatalysts Prepared with Thermomyces lanuginosus Lipase and Different Silica Precursors, Dried using Aerogel and Xerogel Techniques

The use of lipases in industrial processes can result in products with high levels of purity and at the same time reduce pollutant generation and improve both selectivity and yields. In this work, lipase from Thermomyces lanuginosus was immobilized using two different techniques. The first involves the hydrolysis/polycondensation of a silica precursor (tetramethoxysilane (TMOS)) at neutral pH and ambient temperature, and the second one uses tetraethoxysilane (TEOS) as the silica precursor, involving the hydrolysis and polycondensation of the alkoxide in appropriate solvents. After immobilization, the enzymatic preparations were dried using the aerogel and xerogel techniques and then characterized in terms of their hydrolytic activities using a titrimetric method with olive oil and by the formation of 2-phenylethyl acetate in a transesterification reaction. The morphological properties of the materials were characterized using scanning electron microscopy, measurements of the surface area and pore size and volume, thermogravimetric analysis, and exploratory differential calorimetry. The results of the work indicate that the use of different silica precursors (TEOS or TMOS) and different drying techniques (aerogel or xerogel) can significantly affect the properties of the resulting biocatalyst. Drying with supercritical CO2 provided higher enzymatic activities and pore sizes and was therefore preferable to drying, using the xerogel technique. Thermogravimetric analysis and differential scanning calorimetry analyses revealed differences in behavior between the two biocatalyst preparations due to the compounds present.

The novel mesoporous silica aerogel modified with protic ionic liquid for lipase immobilization

Mesoporous silica supports (aerogels) were used to immobilize Burkholderia cepacia lipase (BC) by encapsulation (EN or ENIL), physical adsorption (ADS or ADSIL) and covalent binding (CB or CBIL) into or onto the aerogel modified with protic ionic liquid (PIL). Yield immobilization (Ya) and operational stability were determined by the hydrolytic reaction of olive oil. Ya (37% to 83% by physical adsorption) and operational stability (2 to 23 batches by encapsulation) increased when the support was modified with PIL. For immobilized derivates observed by the BET method, in this case ADS and CB for ADSIL and CBIL, increased pores size was observed, possibly due to the higher amount of BC immobilized conferring Ya and operational stability. This effect was probably attributed to the entry of the enzyme into the pores of the silica aerogel structure. SEM images showed a change in the structure and properties of immobilized lipase derived with PIL. A characteristic FTIR band was obtained for the silanol groups and amides I, IV and V, demonstrating the efficiency of immobilization of BC. The most efficient biocatalysts were ADSIL with regard to yield immobilization and ENIL for operational stability.

CHAPTER 8:Ionic Liquids as Efficient Tools for the Purification of Biomolecules and Bioproducts from Natural Sources

The research on the isolation and recovery of added-value products from biomass has been a hot topic of research in the framework of the biorefinery, aiming a sustainable conversion of biomass into energy, fuels and chemicals. However, the use of the biomass potential is still restricted by the lack of cost-effective processes. In the past decade, ionic liquids (ILs) have been largely investigated as alternative solvents for the extraction and purification of added-value compounds, such as alkaloids, flavonoids, terpenoids and lipids, among others, present in natural sources. Although several works have been published highlighting the potential of ILs as alternative solvents, the isolation/purification of the target compounds as well as the recovery of the solvents for further use has been seldom studied. This chapter presents an overview on the use of supported ILs onto silica or polymers and on IL-based aqueous biphasic systems (ABS) for the purification of high-value compounds present in natural matrices is outlined. The recovery procedures applied to ILs envisaging their recyclability are also presented and discussed. Only after the development of suitable and integrated methodologies, either for the added-value products or ILs recovery, can these be applied in a large scale under the biorefinery framework while decreasing the cost and the environmental footprint of the whole process.

Potential of aqueous two-phase systems for the separation of levodopa from similar biomolecules: Potential of aqueous two-phase systems for the separation

Background Levodopa is a precursor of several neurotransmitters, such as dopamine, and is used in the treatment of the Parkinsons disease. In this work, an alternative strategy was studied to separate levodopa from similar biomolecules using aqueous two-phase systems (ATPS). Results Ternary ATPS composed of polyethylene glycol (PEG) 400 or ionic liquids (ILs), citrate buffer (K3C6H5O7/C6H8O7) at pH 7.0 and water, and quaternary ATPS composed of PEG 400, K3C6H5O7/C6H8O7 at pH 7.0, water and the same ILs at 5 wt%, were studied. The respective liquid-liquid phase diagrams were determined at 298 K to appraise the mixture compositions required to form two-phase systems, followed by studies of the partition of levodopa and structurally similar biomolecules (dopamine, L-phenylalanine, and L-tyrosine). Their partition coefficients and extraction efficiencies have been determined, and the selectivity of the ATPS to separate levodopa from the remaining biomolecules evaluated. Conclusion The results obtained indicated that PEG-based ATPS were the most effective to separate levodopa from L-phenylalanine while the separation from the other biomolecules was better using IL-based ATPS, in particular those based on [P4444]Cl and [N4444]Cl, with extraction efficiencies of levodopa to the salt-rich phase ranging between 62.7 and 74.0%, and of the remaining biomolecules to polymer/IL-rich phase up to 91.5%.

Lipase Immobilization on Silica Xerogel Treated with Protic Ionic Liquid and its Application in Biodiesel Production from Different Oils

Treated silica xerogel with protic ionic liquid (PIL) and bifunctional agents (glutaraldehyde and epichlorohydrin) is a novel support strategy used in the effective immobilization of lipase from Burkholderia cepacia (LBC) by covalent binding. As biocatalysts with the highest activity recovery yields, LBC immobilized by covalent binding with epichlorohydrin without (203%) and with PIL (250%), was assessed by the following the hydrolysis reaction of olive oil and characterized biochemically (Michaelis–Menten constant, optimum pH and temperature, and operational stability). Further, the potential transesterification activity for three substrates: sunflower, soybean, and colza oils, was also determined, achieving a conversion of ethyl esters between 70 and 98%. The supports and the immobilized lipase systems were characterized using Fourier transform infrared spectra (FTIR), scanning electron microscopy (SEM), elemental analysis, and thermogravimetric (TG) analysis.

High concentration solubility and stability of ɛ-poly-l-lysine in an ammonium-based ionic liquid: A suitable media for polypeptide packaging and biomaterial preparation

Packaging of structurally sensitive biomolecules such as proteins, peptides and DNA in non-aqueous media at ambient conditions with chemical and structural stability is important to explore the potential of such biomacromolecules as substrate for functional biomaterial design and for biotechnological applications. In this perspective, solubility, chemical and structural stability of ɛ-poly-l-lysine (ɛ-PL), a homopolypeptide produced by Streptomyces albulus in different ionic liquids (ILs) namely 2-hydroxyethyl ammonium formate (2-HEAF), 2-hydroxyethyl ammonium acetate (2-HEAA), choline formate (Ch-Formate) and choline acetate (Ch-Acetate) was studied. Maximum solubility (15% w/v) of the homopolypeptide was observed in 2-HEAF and lowest was found in Ch-Formate (2% w/v). After regeneration of the dissolved polypeptide in the IL, the IL could be recycled and reused in the dissolution process. Unlike in other ILs, 3-15% w/v of ɛ-PL in 2-HEAF gave formation of a thixotropic thermoreversible soft gel. Molecular docking studies established favourable interactions of [2-HEA]+ cation over [Ch]+ with ɛ-PL indicating [2-HEA]+ as the most promising cation for the dissolution process. However, the role of the anions was also found to be important, which could lead to improvement in polypeptide solubility when combined to the selected cation. The findings demonstrate suitability of the ionic liquids for functionalization of polypeptides for biomaterial preparation.

Enhanced separation performance of aqueous biphasic systems formed by carbohydrates and tetraalkylphosphonium- or tetraalkylammonium-based ionic liquids

Aiming at establishing more effective and sustainable separation processes, herein we propose the use of carbohydrates combined with tetralkylphosphonium- and tetralkylammonium-based ionic liquids (ILs) to form aqueous biphasic systems (ABS). The formation of ABS composed of non-aromatic and non-fluorinated ILs with carbohydrates is here shown for the first time. These novel systems are competive extraction platforms when compared against more conventional ABS formed by ILs and salts or fluorinated ILs and carbohydrates. Finally, it is shown that these systems can be efficiently recovered and reused.