Ionic liquids for aqueous two-phase extraction and stabilization of enzymes

The ionic liquid (IL) Ammoeng110 contains cations with oligoethyleneglycol units and was found to be highly effective for the formation of aqueous two-phase systems (ATPS) that can be used for the biocompatible purification of active enzymes. Above critical concentrations of the IL and an inorganic salt in aqueous solution, phase separation takes place resulting in the formation of an IL-enriched upper and a salt-enriched lower phase. For the optimization of the composition of IL-based ATPS with regard to the extraction of catalytically active enzymes, the Box-Wilson method of experimental design was successfully applied; IL-based ATPS proved to be suitable for the purification and stabilization of two different alcohol dehydrogenases (from Lactobacillus brevis and a thermophilic bacterium). Both enzymes were enriched in the IL-containing upper phase resulting in an increase of specific activity by a factor of 2 and 4 respectively. Furthermore, the presence of IL within the system provided the opportunity to combine the extraction process with the performance of enzyme-catalyzed reactions. The IL was found to exhibit a stability improving effect on both enzymes and a solubility enhancing effect on hydrophobic substrates. Thus the conversion and volumetric productivity of ADH catalyzed reduction of acetophenone could be increased significantly.     

Design of ionic liquids for lipase purification

Aqueous two-phase systems (ATPS) are considered as efficient downstream processing techniques in the production and purification of enzymes, since they can be considered harmless to biomolecules due to their high water content and due to the possibility of maintaining a neutral pH value in the medium. A recent type of alternative ATPS is based on hydrophilic ionic liquids (ILs) and salting-out inducing salts. The aim of this work was to study the lipase (Candida antarctica lipase B - CaLB) partitioning in several ATPS composed of ionic liquids (ILs) and inorganic salts, and to identify the best IL for the enzyme purification. For that purpose a wide range of IL cations and anions, and some of their combinations were studied. For each system the enzyme partitioning between the two phases was measured and the purification factors and enzyme recoveries were determined. The results indicate that the lipase maximum purification and recovery were obtained for cations with a C(8) side alkyl chain, the [N(CN)(2)] anion and ILs belonging to the pyridinium family. However, the highest purification parameters were observed for 1-methyl-3-octylimidazolium chloride [C(8)mim]Cl, suggesting that the IL extraction capability does not result from a cumulative character of the individual characteristics of ILs. The results indicate that the IL based ATPS have an improved performance in the lipase purification and recovery.     

Impact of cell disruption and polymer recycling upon aqueous two-phase processes for protein recovery

A practical study is presented of the influence of cell debris and polymer recycling upon the operation of two-stage acqueous two-phase systems (ATPS) for the recovery of yeast bulk protein, pyruvate kinase and fumarase. Brewers' yeast was disrupted using one of two types of high-pressure homogenisers or a bead mill. The different cell debris suspensions were partitioned in a single PEG-phosphate ATPS extraction and the efficiency of solid-liquid separation was examined. A continuously operated two-stage ATPS process, using spray columns, is presented and practical problems of polymer recycling are discussed. Conclusions are drawn concerning the generic implementation and operational stability of ATPS in practical protein recoveries.     

PEG–salt aqueous two-phase systems: an attractive and versatile liquid–liquid extraction technology for the downstream processing of proteins and enzymes

Nowadays, there is an increasing demand to establish new feasible, efficient downstream processing (DSP) techniques in biotechnology and related fields. Although several conventional DSP technologies have been widely employed, they are usually expensive and time-consuming and often provide only low recovery yields. Hence, the DSP is one major bottleneck for the commercialization of biological products. In this context, polyethylene glycol (PEG)–salt aqueous two-phase systems (ATPS) represent a promising, efficient liquid–liquid extraction technology for the DSP of various biomolecules, such as proteins and enzymes. Furthermore, ATPS can overcome the limitations of traditional DSP techniques and have gained importance for applications in several fields of biotechnology due to versatile advantages over conventional DSP methods, such as biocompatibility, technical simplicity, and easy scale-up potential. In the present review, various practical applications of PEG–salt ATPS are presented to highlight their feasibility to operate as an attractive and versatile liquid–liquid extraction technology for the DSP of proteins and enzymes, thus facilitating the approach of new researchers to this technique. Thereby, single- and multi-stage extraction, several process integration methods, as well as large-scale extraction and purification of proteins regarding technical aspects, scale-up, recycling of process chemicals, and economic aspects are discussed.     

Lipase-catalyzed glycerolysis in ionic liquids directed towards diglyceride synthesis

This work examined the lipase-catalyzed glycerolysis of triglycerides (TG) in a list of commercially available ionic liquids (ILs) with varied cations and anions for the purpose of developing an efficient reaction protocol for diglyceride (DG) production and to understand whether ILs could assist the reaction systems. The reaction performances (reaction rate, TG conversion and DG yield) were found to be greatly dependent on the structure and property of ILs. The reactions in [TOMA·Tf₂N] and Ammoeng 120 produced comparable yield of DG to those most efficient conventional systems but with less by-products. Temperature enhancement generally yields positive effect on the conversion of TG, which was much more significant for the ILs with high viscosity. Unusually, increasing substrate concentration in many types of ILs led to enhanced conversion and yield; whereas the increase of glycerol/TG ratio resulted in a dramatic improvement of the reactions in the ILs with strong acidic anions. This work also sorted out some promising IL candidates, namely the ILs with good DG formation selectivity and the ones being able to achieve high TG conversion, which offered possibility to design binary IL systems. Overall, this study presented the first attempt concerning evaluation and characterization of lipase-catalyzed glycerolysis of TG for DG production in IL-based systems.     

Membrane interactions of ionic liquids: Possible determinants for biological activity and toxicity

Ionic liquids (ILs) are a class of diverse organic salts with relatively low melting points (below 100°C) which have attracted considerable interest as a promising "green" substitute for organic solvents. The broad solvation properties of ILs and their high solubility in water, however, present health risks, in particular since it was shown that many ILs exhibit cytotoxic properties. In this context, interactions of ILs with the cellular membrane are believed to constitute a primary culprit for toxicity. We present a comprehensive biophysical and microscopy study of membrane interactions of a series of ILs having different side-chain compositions and lengths, and cationic head-group structures and orientations. The experimental data reveal that the ILs studied exhibit distinct mechanisms of membrane binding, insertion, and disruption which could be correlated with their biological activities. The results indicate, in particular, that both the side chain composition and particularly the head-groups of ILs constitute determinants for membrane activity and consequent cell toxicity. This work suggests that tuning membrane interactions of ILs should be an important factor for designing future compounds with benign environmental impact.     

Aqueous two-phase systems

Biphasic systems formed by mixing of two polymers or a polymer and a salt in water can be used for separation of cells, membranes, viruses, proteins, nucleic acids, and other biomolecules. The partitioning between the two phases is dependent on the surface properties and conformation of the materials, and also on the composition of the two-phase system. The mechanism of partitioning is, however, complex and not easily predicted. Aqueous two-phase systems (ATPS) have proven to be a useful tool for analysis of biomolecular and cellular surfaces and their interactions, fractionation of cell populations, product recovery in biotechnology, and so forth. Potential for environmental remediation has also been suggested. Because ATPS are easily scalable and are also able to hold high biomass load in comparison with other separation techniques, the application that has attracted most interest so far has been the large-scale recovery of proteins from crude feedstocks. As chemicals constitute the major cost factor for large-scale systems, use of easily recyclable phase components and the phase systems generated by a single-phase chemical in water are being studied.     

Recent advances in exploiting ionic liquids for biomolecules: Solubility,stability and applications

The technological utility of biomolecules (e.g. proteins, enzymes and DNA) can be significantly enhanced by combining them with ionic liquids (ILs) – potentially attractive ”green“ and ”designer“ solvents – rather than using in conventional organic solvents or water. In recent years, ILs have been used as solvents, cosolvents, and reagents for biocatalysis, biotransformation, protein preservation and stabilization, DNA solubilization and stabilization, and other biomolecule‐based applications. Using ILs can dramatically enhance the structural and chemical stability of proteins, DNA, and enzymes. This article reviews the recent technological developments of ILs in protein‐, enzyme‐, and DNA‐based applications. We discuss the different routes to increase biomolecule stability and activity in ILs, and the design of biomolecule‐friendly ILs that can dissolve biomolecules with minimum alteration to their structure. This information will be helpful to design IL‐based processes in biotechnology and the biological sciences that can serve as novel and selective processes for enzymatic reactions, protein and DNA stability, and other biomolecule‐based applications.     

Efficient pretreatment of lignocellulose in ionic liquids/co-solvent for enzymatic hydrolysis enhancement into fermentable sugars

Ionic liquids (ILs) have been widely used as alternative solvents for biomass pretreatment, however, efficient methods that enable economically use of ILs at large scale have not been established. In this study, a new method in which ILs and polar organic solvents (ILs/co-solvent systems) was proposed for efficient pretreatment of lignocellulosic materials. The combination use of appropriate ILs and organic co-solvents can significantly enhance the solubility of lignocellulose due to the lower viscosity of ILs/co-solvent mixture as compared to those of pure ILs while the hydrogen bond basicity was maintained. In addition, the solubility of lignocellulosic materials in ILs/co-solvent system was found to be correlated with the Kamlet-Taft solvent parameters. Moreover, the use of microwave heating also enhances the efficiency of lignocellulose pretreatment. For example, the microwave-assisted [Emim][OAc]-DMSO (1:1 volume ratio) treated-rice straw could be hydrolyzed at least 22 times faster than that of untreated-rice straw by cellulase from Trichoderma reesei. This enhancement was attributed by several factors including more efficient lignin extraction, less crystalline cellulose and lower residual ILs in treated-rice straw. The produced sugars can be effectively fermented by Pichia stipitis for ethanol production. Moreover, [Emim][OAc]-DMSO mixture could be reused at least 5 times without significantly decrease in effectiveness demonstrated that the use of ILs/co-solvent was potential alternative method for large-scale biomass pretreatment.     

Ionic liquid-based in situ product removal design exemplified for an acetone–butanol–ethanol fermentation

Selecting an appropriate separation technique is essential for the application of in situ product removal (ISPR) technology in biological processes. In this work, a three-stage systematic design method is proposed as a guide to integrate ionic liquid (IL)-based separation techniques into ISPR. This design method combines the selection of a suitable ISPR processing scheme, the optimal design of an IL-based liquid–liquid extraction (LLE) system followed by process simulation and evaluation. As a proof of concept, results for a conventional acetone–butanol–ethanol fermentation are presented (40,000 ton/year butanol production). In this application, ILs tetradecyl(trihexyl)phosphonium tetracyanoborate ([TDPh][TCB]) and tetraoctylammonium 2-methyl-1-naphthoate ([TOA] [MNaph]) are identified as the optimal solvents from computer-aided IL design (CAILD) method and reported experimental data, respectively. The dynamic simulation results for the fermentation process show that, the productivity of IL-based in situ (fed-batch) process and in situ (batch) process is around 2.7 and 1.8fold that of base case. Additionally, the IL-based in situ (fed-batch) process and in situ (batch) process also have significant energy savings (79.6% and 77.6%) when compared to the base case.     

Aqueous two-phase fractionation of biological suspensions for protein recovery from bovine blood

The practical application of a two-stage aqueous two-phase systems (ATPS) for the fractionation and recovery of proteins from biological suspensions is described. A model process for the recovery of proteins from whole bovine blood was selected to study the implementation of an ATPS process. Recycling of used PEG into the initial extraction stage did not significantly influence the partition behaviour of serum albumin in subsequent cycles. © Rapid Science Ltd. 1998     

Extraction of tetracycline from fermentation broth using aqueous two-phase systems composed of polyethylene glycol and cholinium-based salts

Aiming at developing not only cheaper but also biocompatible and sustainable extraction and purification processes for antibiotics, in this work it was evaluated the ability of aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and cholinium-based salts to extract tetracycline from the fermented broth of Streptomyces aureofaciens. Conventional polymer/salt and salt/salt ATPS were also studied for comparison purposes. The novel systems here proposed are able to extract tetracycline directly from the fermentation broth with extraction efficiencies higher than 80%. A tailored extraction ability of these systems can also be achieved, with preferential extractions either for the polymer- or salt-rich phases, and which further depend on the cholinium-based salt employed. The gathered results support the applicability of biocompatible ATPS in the extraction of antibiotics from complex matrices and can be envisaged as valuable platforms to be applied at the industrial level by pharmaceutical companies.     

Determination of the active constituents in Arnebia euchroma (Royle) Johnst. by ionic liquid-based ultrasonic-assisted extraction high-performance liquid chromatography

Shikonin and β,β'-dimethylacrylshikonin in Arnebia euchroma (Royle) Johnst. were extracted by ionic liquid-based ultrasonic-assisted extraction (IL-based UAE) and determined by high-performance liquid chromatography (HPLC). The dried powder of A. euchroma (Royle) Johnst. was mixed with a room temperature ionic liquid [C(6)MIM][BF(4)] to form a suspension, and then the ultrasonic extraction was performed in a water bath at ambient temperature. The calibration curve showed good linear relationship (r>0.9998) in the concentration range of 1.75-140 μg/mL for shikonin and 2.15-1360 μg/mL for β,β'-dimethylacrylshikonin. The recoveries were between 69.79% and 82.35%. The IL-based UAE is free of volatile organic solvents, and consumes less sample, time and solvent, compared with regular ultrasonic and Soxhlet extraction. There was no obvious difference in the extraction yields of active constitutions obtained by the three extraction methods.     

The potential application of aqueous two-phase systems for in situ recovery of 6-pentyl-infinity-pyrone produced by Trichoderma harzianum

Commercial production of aroma compounds by de novo microbial biosynthesis has been principally limited by the low productivity so far achieved. Production of 6-pentyl-alpha-pyrone (6PP), a coconut-like aroma compound, by Trichoderma harzianum has been limited by the toxic effect that occurs even at low concentration (<100 ppm). This work evaluated the feasibility of the use of aqueous-two phase systems (ATPS), as in situ extraction systems, in order to overcome the toxic effects of 6PP and to improve culture productivity. The partition behaviour of 6-pentyl-alpha-pyrone and Trichoderma harzianum mycelium in polyethylene glycol (PEG)-salt and PEG-dextran two-phase systems was investigated and it is reported for the first time. The evaluation of system parameters such as PEG molecular mass, concentration of PEG as well as salt, volume ratio (Vr) and dextran molecular mass, was carried out to determine under which conditions the 6PP partitions to the opposite phase that mycelium does. PEG-dextran systems proved to be unsuitable for the in situ recovery of 6PP because either 6PP and biomass partitioned to the same phase or a large extraction phase was required for the process. ATPS extraction comprising Vr = 0.26, PEG 1450 (7.2% w/w) and sulphate (16.6% w/w) provided the best conditions for the maximum accumulation of the biomass into the bottom phase and concentrated the 6PP in the opposite phase (i.e. 86% of biomass and 56% of 6PP of the total amount loaded from the fermentation extract into the ATPS) for ex situ bioseparation. However, this system caused complete inhibition of the growth of the microorganism during the in situ bioseparation, probably as a consequence of the high ionic strength resulting from the salt concentration. Consequently, two ATPS PEG 8000-sulphate (12%/7% and 6%/14%) were evaluated and proved to be more suitable in the potential application for the in situ recovery of 6PP.     

Protein purification of Arthrospira platensis using aqueous two-phase system composed of polyethylene glycol and potassium phosphate/sodium citrate

Journal of Applied Phycology - Aqueous two-phase systems (ATPS) stand out as an alternative technique for recovering and concentrating proteins. However, the study of ATPS to recover Arthrospira...     

Effect of ionic liquids,organic solvents and supercritical CO2 pretreatment on the conformation and catalytic properties of Candida rugosa lipase

The objective of this work is to assess the structure and activity of Candida rugosa lipase (CRL) pretreated with seventeen ionic liquids (ILs), five organic solvents and super-critical carbon dioxide (SC-CO₂). The results revealed that anion selection of ILs showed generally much greater effects on CRL esterification activity than cation choice, and CRL pretreated by ILs with strong water miscible properties showed very low esterification activity. The highest CRL activity treated with ILs [Hmim][PF6] was obtained with the value of 45078.0 U/g-protein. Furthermore, the CRL activities pretreated with five conventional organic solvents were also examined and the values increased with the log P decrease of organic solvents when log P was lower than 2.0. Finally, the CRL activities were respectively 1.2- and 1.3-fold higher over the untreated ones after pretreatment with sub- and super-critical CO₂ under the pressures of 6 MPa and 15 MPa at 40 °C for 20 min. Further analyses via FT-IR demonstrated that the high activity of CRL pretreated with ILs, organic solvents and SC-CO₂ was probably caused by the changes of CRL secondary structure. In conclusion, the results in this work will be helpful for us to choose the suitable reaction medium in CRL biocatalysis and biotransformation reactions.     

Recovery of laccase from the residual compost of Agaricus bisporus in aqueous two-phase systems

The potential use of aqueous two-phase systems (ATPS) to establish a viable protocol for the recovery of laccase from the residual compost of Agaricus bisporus was evaluated. The evaluation of system parameters such as poly (ethylene glycol) (PEG) molecular mass, concentration of PEG as well as salt and system pH was carried out to determine under which conditions the laccase concentrates predominantly to the top PEG-rich phase. PEG 1000–phosphate ATPS proved to be suitable for the primary recovery of laccase. An extraction ATPS stage comprising volume ratio equal to 1.0, PEG 1000 18.2% (w/w), phosphate 15.0% (w/w), system pH of 7.0 and loaded with 5% (w/w) of crude extract from residual compost allowed the laccase recovery. The use of ATPS resulted in one-single primary recovery stage process that produced an overall yield of 95%. The results reported here demonstrated the potential application of ATPS for the valorisation of residual material and the potential establishment of a downstream process to obtain value added products with commercial application.     

Purification of (S)-3-cyano-5-methylhexanoic acid from bioconversion broth using an acetone/ammonium sulfate aqueous two-phase system

(S)-3-Cyano-5-methylhexanoic acid ((S)-CMHA) is the key chiral intermediate of pregabalin. In this paper, an aqueous two-phase system (ATPS) was developed to extract (S)-CMHA from nitrilase-catalyzed bioconversion broth. Inorganic salts and hydrophilic solvents were screened to form ATPS, among which an acetone/ammonium sulfate ATPS was investigated in detail, including phase diagram, effect of phase composition and stability of (S)-CMHA. The maximum product recovery of 99.15% was obtained by an optimized ATPS system composed of 15% (w/w) ammonium sulfate and 35% (w/w) acetone with the removal of 99% cells and 86.27% proteins. The total (S)-CMHA yield reached 92.11% after back-extraction. The recycling use of ammonium sulfate was investigated, and 93.10% of salt in the salt-rich phase was recovered with the addition of methanol. The results demonstrated the efficiency of the two-step extraction process for separation of (S)-CMHA.     

Response surface methodology for the extraction of wedelolactone from Eclipta alba using aqueous two-phase extraction

Abstract

Aqueous two-phase extraction of wedelolactone from Eclipta alba was studied using the polymer-salt system. The system consisted of polyethylene glycol (PEG) as a top phase (polymer) and sodium citrate as a bottom phase (salt). Process parameters such as PEG concentration, PEG molecular weight, salt concentration, and pH have been optimized using response surface methodology (RSM) with the help of central composite design (CCD). The optimized conditions for aqueous two-phase system (ATPS), in the case of one factor at a time approach, were found as PEG 6000, PEG concentration 18% (w/v), salt concentration 16% (w/v), and pH 7; with maximum extraction yield of 6.52?mg/g. While, RSM studies showed maximum extraction yield of 6.73?mg/g with the optimized parameters as PEG 6000, PEG concentration 18% (w/v), salt concentration 17.96% (w/v), and pH 7. ATPS was found to give a 1.3 fold increase in the extraction yield of wedelolactone as compared to other conventional extraction methods.     

Aqueous two-phase systems for biotechnical use.

The different kinds of aqueous two-phase systems for accepted or potential use in biotechnology are summarized. Some properties of interest for the extractive use are discussed.