Use Of Aqueous Two-phase Systems Research Articles

Recovery of crocins from saffron stigmas (Crocus sativus) in aqueous two-phase systems

Crocins are carotenoid derivates that have recently attracted the interest of the scientific community due to their nutraceutical properties. Saffron (dry Crocus sativus stigmas) is one of the main known sources of crocins. In this study the potential use of aqueous two-phase system (ATPS) for the extraction of crocins from C. sativus stigmas was evaluated. The partitioning behavior of crocins in different types of ATPS (polymer–polymer, polymer–salt, alcohol–salt and ionic liquid–salt) was evaluated. Ethanol–potassium phosphate ATPS were selected based on their high top phase recovery yield and low cost of system constituents. The evaluation and optimization of system parameters rendered conditions (VR=3.2, ethanol 19.8% (w/w), potassium phosphate 16.5% (w/w), TLL of 25% (w/w), 0.1M NaCl and 2% (w/w) of sample load) under which more than 75% of total crocins were recovered in the top (ethanol rich) phase, whereas the wasted stigmas accumulated in the bottom phase. Lastly, a comparison between an optimized solid–liquid extraction using ethanol:water as solvent and ATPS was conducted demonstrating that similar yields are achieved with both strategies (76.89±1.8% and 79.27±1.6%, respectively). However, ATPS rendered a higher extraction selectivity of 1.3±0.04mg of crocins for each mg of phenolic compound, whereas ethanolic extraction showed a selectivity of 0.87±0.01. The results reported herein demonstrate the potential application of ATPS, particularly ethanol–potassium phosphate systems, for the recovery of crocins from C. sativus stigmas.

Application of Aqueous Two‐Phase Systems for the Potential Extractive Fermentation of Cyanobacterial Products

AbstractThe potential use of aqueous two‐phase systems (ATPS) to establish a viable protocol for the in situ recovery of cyanobacterial products was evaluated. The evaluation of system parameters such as poly (ethylene glycol) (PEG) molecular mass, concentration of PEG and salt was carried out to determine the conditions under which Synechocystis sp. PCC 6803 cell and cyanobacterial products, i.e., β‐carotene and lutein, become concentrated in opposite phases. PEG‐phosphate ATPS proved to be unsuitable for the recovery of cyanobacterial products due to the negative effect of the salt upon the cell growth. The use of ATPS PEG‐dextran (6.6 % w/w PEG 3350, 8.4 % w/w dextran 66900, TLL 17.3 % w/w, VR 1.0, pH 7) and (4.22 % w/w PEG 8000, 9.77 % w/w dextran 66900, TLL 18 % w/w, VR 1.0, pH 7) resulted in the growth of cyanobacteria (Synechocystis sp. PCC 6803) and the concentration of lutein in opposite phases. However, β‐carotene was seen to concentrate in the top phase together with the biomass. The results reported here demonstrate the potential application of ATPS to establish the conditions for an extractive fermentation prototype process for the recovery of cyanobacterial products.

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.

The application of aqueous two-phase systems to oligosaccharide synthesis by alpha-mannosidase catalysed glycosyl transfer reactions.

Aqueous two-phase systems formed from PEG and dextran have been applied to the synthesis of oligosaccharide by Jack bean alpha-mannosidase in reverse. Whilst rates of synthesis and percentage yields were similar in two-phase systems and one-phase aqueous buffer systems, a ten-fold increase in yield of product per unit of enzyme was seen. In addition, the use of aqueous two-phase systems offers potential process advantages over one-phase systems for the synthesis of oligosaccharides.

Aggregation and denaturation of antibodies: a capillary electrophoresis, dynamic light scattering, and aqueous two-phase partitioning study

Protein denaturation and aggregation are well-known problems in the pharmaceutical industry. As the protein aggregates, it loses its biological activity and creates problems in its administration to patients. In this paper, we explore the use of aqueous two-phase systems, capillary zone electrophoresis, and dynamic light scattering for the monitoring of protein denaturation and aggregation. Our studies focus on human IgG and HSA. Capillary zone electrophoresis was used to monitor changes in the charge to size ratio of the proteins upon denaturation and dynamic light scattering was used to detect the presence of any aggregates and to monitor the size of the proteins. The information obtained from aqueous two-phase partitioning is similar to that obtained from capillary zone electrophoresis. The simplicity of aqueous two-phase system and its low cost (compared to the other analytical techniques) suggest that it can be routinely used for the quality control of some pharmaceutical preparations.

Recovery in aqueous two-phase systems of lutein produced by the green microalga Chlorella protothecoides

In this study the potential use of aqueous two-phase system (ATPS) to establish a viable process for the recovery of lutein from the green microalga Chlorella protothecoides is evaluated. The partitioning behaviour of lutein, a representative model of natural compounds of commercial interest, was investigated in a polyethylene glycol (PEG)-phosphate system. An evaluation of system parameters including PEG molecular mass, the concentrations of PEG, phosphate and product concentration was conducted, to estimate conditions under which lutein partitions preferentially to the top phase whilst cell debris partition to the opposite phase. The necessary addition of ethanol to the ATPS for the dissolution of lutein affected the phase formation and such effect was evaluated using the change in the volume ratio produced. ATPS extraction comprising Vr=1.0, PEG 8000 22.9% (w/w) and phosphate 10.3% (w/w), pH 7.0 provided the conditions for the concentration of lutein into the upper phase and the cell debris preferentially to the bottom phase. The use of ATPS resulted in a primary recovery process to obtain lutein with an overall product yield of 81.0±2.8%. The findings reported here demonstrate the potential of ATPS for the further development of a prototype process to recover lutein from C. protothecoides as a first step for the generic application of this technique.

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.

Aroma compounds recovery from mycelial cultures in aqueous two-phase processes

This paper presents the evaluation of the potential use of aqueous two-phase systems (ATPS) for the recovery of 6-pentyl-α-pyrone (6PP) produced by Trichoderma harzianum. The partition behaviour of 6PP and Trichoderma harzianum mycelium (biomass) in polyethylene glycol (PEG)–salt (phosphate and sulphate) and PEG–dextran ATPS was investigated. The influence of defined system parameters (e.g. molecular mass of PEG and dextran, volume ratio, etc.) on the partition behaviour of 6PP and Trichoderma harzianum mycelium was evaluated to select under which conditions 6PP and mycelium partition to opposite phases. In PEG–dextran systems either large extraction phases were required or mycelium and 6PP partitioned to the same phase. ATPS comprising V r=0.23, PEG 8000 6.6% w/w and sulphate 14.0% w/w provided the best conditions to satisfy the process requirement of biomass accumulation into the bottom phase and 6PP concentration in the top phase.

Liquid-liquid extraction of biomolecules in downstream processing - A review paper

Economic analysis shows that protein separation and purification are a very important aspect of biomolecules production and processing. This is particularly true for protein processing which, because of the complexity of the starting material, often requires many steps to reach the levels of purity required for medical and food applications. The separation specialists' task is to develop safe and simple processes to achieve products with a high level of purity. On a large scale, chromatography of proteins is not an easily applied method, although on a laboratory scale it is very effective and relatively simple. When it is scaled up, shortcomings such as discontinuity in the process, slow protein diffusion and large pressure drops in the system are seen. For these reasons a substantial research effort has been directed toward the use of aqueous two-phase systems (ATPSs) to replace the initial steps in protein purification and chromatography. This article reviews the chronology and main ATPS fundamentals and discuss the broader applications of this type of system in the extraction and separation of biomolecules.

Use of aqueous two-phase systems for in situ extraction of water soluble antibiotics during their synthesis by enzymes immobilized on porous supports

Yields of kinetically controlled synthesis of antibiotics catalyzed by penicillin G acylase from Escherichia coli (PGA) have been greatly increased by continuous extraction of water soluble products (cephalexin) away from the surroundings of the enzyme. In this way its very rapid enzymatic hydrolysis has been avoided. Enzymes covalently immobilized inside porous supports acting in aqueous two-phase systems have been used to achieve such improvements of synthetic yields. Before the reaction is started, the porous structure of the biocatalyst can be washed and filled with one selected phase. In this way, when the pre-equilibrated biocatalyst is mixed with the second phase (where the reaction product will be extracted), the immobilized enzyme remains in the first selected phase in spite of its possibly different natural trend. Partition coefficients (K) of cephalexin in very different aqueous two-phase systems were firstly evaluated. High K values were obtained under drastic conditions. The best K value for cephalexin (23) was found in 100% PEG 600-3 M ammonium sulfate where cephalexin was extracted to the PEG phase. Pre-incubation of immobilized PGA derivatives in ammonium sulfate and further suspension with 100% PEG 600 allowed us to obtain a 90% synthetic yield of cephalexin from 150 mM phenylglycine methyl ester and 100 mM 7-amino desacetoxicephalosporanic acid (7-ADCA). In this reaction system, the immobilized enzyme remains in the ammonium sulfate phase and hydrolysis of the antibiotic becomes suppressed because of its continuous extraction to the PEG phase. On the contrary, synthetic yields of a similar process carried out in monophasic systems were much lower (55%) because of a rapid enzymatic hydrolysis of cephalexin.

Use of aqueous two-phase systems in the purification of human interferon-α1 from recombinant Escherichia coli

Intracellular human recombinant interferon α1 (rhIFN-α1) has been purified using two consecutive aqueous two-phase extractions: selective extraction followed by back extraction. In the selective extraction step using polyethylene glycol (PEG)-potassium phosphate aqueous two-phase systems, optimization of the systems was achieved by varying the concentrations of PEG-phosphate ester, phosphate, added inorganic salts, pH, and biomass load. The optimum condition was found to be 22% (w/w) synthesized PEG-phosphate ester, 16% (w/w) potassium phosphate and 3% (w/w) NaCl at pH 6.9 with 10% (wet w/w) biomass load. The corresponding top phase yield of rhIFN-α1 was 99.6% with a purification factor of 25. It was observed that most of the modified PEG is partitioned to the phosphate-enriched bottom phase, so the mechanism for enhancement to the extraction is likely to be an electrostatic expulsion effect. In the back extraction step where the partially purified IFN is transferred from the PEG-rich top phase to a phosphate buffer, the concentrations of PEG-phosphate ester and phosphate, pH, and other parameters have been adjusted. The pH was found to be the most sensitive. A back extraction system using 20% (w/w) PEG-phosphate ester and 10% (w/w) potassium phosphate system at pH 6.0 gives the yield of rhIFN-α1 in the bottom phase (approximately 76%). This newly established back extraction procedure avoids the use of a cumbersome gel chromatography while the yield achieved is fairly high.

Liquid-liquid partitioning of some enzymes, especially phosphofuctokinase, from Saccharomyces cerevisiae at subzero temperature

The effects of low temperature (−18°C) on the stability and partitioning of some glycolytic enzymes within an aqueous two-phase system were studied. The enzymes were phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase present in a crude extract of bakers' yeast. The partitioning of pure phosphofructokinase, isolated from bakers' yeast, was also examined. The two-phase systems were composed of water, poly(ethylene glycol), dextran, and ethylene glycol and buffer. The influence on the partitioning of the presence of ethylene glycol, phenylmethylsulfonyl fluoride and poly(ethylene glycol)-bound Cibacron Blue F3G-A was investigated at −18, 0 and (in some cases) 20°C. The presence of ethylene glycol, phase polymers and low temperature stabilized all three enzyme activities. Cibacron Blue, an affinity ligand for phosphofructokinase, increased its partitioning into the upper phase with decreasing temperature. Depending on the conditions, various amounts of the enzymes were recovered at the interface, also in systems not containing ethylene glycol. The implications of the observed effects on the use of aqueous two-phase systems for the extraction and fractionation of proteins are discussed.

Use of aqueous two-phase systems in sample preparation for polymerase chain reaction-based detection of microorganisms

An aqueous two-phase system, consisting of poly(ethylene glycol) (PEG) and dextran, was employed to separate polymerase chain reaction (PCR)-inhibitory substances from bacterial cells. The PCR inhibition of four soft cheeses was examined and three of them were found to be strongly PCR-inhibitory. Extraction of the PCR-inhibitory soft cheeses inoculated with Listeria monocytogenes in an aqueous two-phase system containing 8% (w/w) PEG 400 and 8% (w/w) dextran 500, was found to lower the PCR detection level of L. monocytogenes by more than four orders of magnitude in two of the cheeses compared to the case where no such sample pretreatment was performed. Depending on the type of cheese used, the PCR-inhibitory factors were found to be enriched in either the top or botton phase in the aqueous two-phase system. These results show that different soft cheeses contain different types and amounts of PCR-inhibitory substances.

Steroid transformation in aqueous two-phase systems: side-chain degradation of cholesterol by Mycobacterium sp.

We report on the use of aqueous two-phase systems for the side-chain degradation of cholesterol into androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) by Mycobacterium sp. A problem encountered at the start of this investigation, extensive aggregation of the cells, was solved by careful choice of the polymer and detergent composition of the phase system. The systems showing the best properties were composed of polyethylene glycol, dextran and Brij 35 or polyvinylpyrrolidone, dextran and Brij 35 (or Tween 40). The bacterium maintained in the upper polyethylene glycol-rich (or polyvinylpyrrolidone-rich) phase was found to have high activity. The substrated partitioned to the upper phase or the interface (if present as crystals), while the products had partition coefficients around 2.

Extractive bioconversion in aqueous two-phase systems

The transformation of hydrocortisone to prednisolone was studied in aqueous two-phase systems, as a model for the extractive bioconversion of fine chemicals. The bacterium, Arthrobacter simplex, was able to grow in the two-phase system and the cells could be revitalized after a period of use. Use of aqueous two-phase systems made it possible to operate the reaction at higher substrate concentrations than in pure buffer solution. An adsorptive method to remove the product from the top phase was tested and shown to be both efficient and compatible with the overall process. In order to reduce the costs of operation in aqueous two-phase systems, a cheaper starch-based polymer, Reppal-PES, was successfully used as a substitute for dextran.

Anomalous partitioning of aryl acylamidase in aqueous two-phase systems

The use of aqueous two-phase systems for the extraction of aryl acylamidase (aryl-acylamide amidohydrolase, EC 3.5.1.13) from cell lysates of Pseudomonas fluorescens has been examined in an attempt to improve the large scale purification of this enzyme. In poly(ethylene glycol)/dextran systems the enzyme from cell lysates behaved anomalously, the partition coefficient increasing with either increasing poly(ethylene glycol) (PEG) concentration or PEG average molecular weight. When partially purified enzyme was used, the normal effect of PEG concentration and molecular weight was observed. However, inclusion of phosphate buffer pH 7.6 in this phase system reproduced the anomalous partitioning behaviour observed in cell lysates. Possible reasons for this behaviour are discussed.