Affinity-enhanced protein partitioning in decyl beta-D-glucopyranoside two-phase aqueous micellar systems

Liquid-liquid extraction in two-phase aqueous complex-fluid systems has been proposed as a scalable, versatile, and cost-effective purification method for the downstream processing of biotechnological products. In the case of two-phase aqueous micellar systems, careful choices of the phase-forming surfactants or surfactant mixtures allow these systems to separate biomolecules based on size, hydrophobicity, charge, or specific affinity. In this article, we investigate the affinity-enhanced partitioning of a model affinity-tagged protei--green fluorescent protein fused to a family 9 carbohydrate-binding module (CBM9-GFP)--in a two-phase aqueous micellar system generated from the nonionic surfactant n-decyl beta-D-glucopyranoside (C10G1), which acts simultaneously as the phase-former and the affinity ligand. In this simple system, CBM9-GFP was extracted preferentially into the micelle-rich phase, despite the opposing tendency of the steric, excluded-volume interactions operating between the protein and the micelles. We obtained more than a sixfold increase (from 0.47 to 3.1) in the protein partition coefficient (Kp), as compared to a control case where the affinity interactions were "turned off" by the addition of a competitive inhibitor (glucose). It was demonstrated conclusively that the observed increase in Kp can be attributed to the specific affinity between the CBM9 domain and the affinity surfactant C10G1, suggesting that the method can be generally applied to any CBM9-tagged protein. To rationalize the observed phenomenon of affinity-enhanced partitioning in two-phase aqueous micellar systems, we formulated a theoretical framework to model the protein partition coefficient. The modeling approach accounts for both the excluded-volume interactions and the affinity interactions between the protein and the surfactants, and considers the contributions from the monomeric and the micellar surfactants separately. The model was shown to be consistent with the experimental data, as well as with our current understanding of the CBM9 domain.     

Glucose-6-phosphate dehydrogenase partitioning in two-phase aqueous mixed (nonionic/cationic) micellar systems

The enzyme glucose-6-phosphate dehydrogenase (G6PD) plays an important role in maintaining the level of NADPH and in producing pentose phosphates for nucleotide biosynthesis. It is also of great value as an analytical reagent, being used in various quantitative assays. In searching for new strategies to purify this enzyme, the partitioning of G6PD in two-phase aqueous mixed (nonionic/cationic) micellar systems was investigated both experimentally and theoretically. Our results indicate that the use of a two-phase aqueous mixed micellar system composed of the nonionic surfactant C(10)E(4) (n-decyl tetra(ethylene oxide)) and the cationic surfactant C(n)TAB (alkyltrimethylammonium bromide, n = 8, 10, or 12) can improve significantly the partitioning behavior of G6PD relative to that obtained in the two-phase aqueous C(10)E(4) micellar system. This improvement can be attributed to electrostatic attractions between the positively charged mixed (nonionic/cationic) micelles and the net negatively charged enzyme G6PD, resulting in the preferential partitioning of G6PD to the top, mixed micelle-rich phase of the two-phase aqueous mixed micellar systems. The effect of varying the cationic surfactant tail length (n = 8, 10, and 12) on the denaturation and partitioning behavior of G6PD in the C(10)E(4) /C(n)TAB/buffer system was investigated. It was found that C(8)TAB is the least denaturing to G6PD, followed by C(10)TAB and C(12)TAB. However, the C(10)E(4)/C(12)TAB/buffer system generated stronger electrostatic attractions with the net negatively charged enzyme G6PD than the C(10)E(4)/C(10)TAB/buffer and the C(10)E(4)/C(8)TAB/buffer systems, when using the same amount of cationic surfactant. Overall, the two-phase aqueous mixed (C(10)E(4)/C(10)TAB) micellar system yielded the highest G6PD partition coefficient of 7.7, with a G6PD yield in the top phase of 71%, providing the optimal balance between the denaturing effect and the electrostatic attractions for the three cationic surfactants examined. A recently developed theoretical framework to predict protein partition coefficients in two-phase aqueous mixed (nonionic/ionic) micellar systems was implemented, and the theoretically predicted G6PD partition coefficients were found to be in reasonable quantitative agreement with the experimentally measured ones.     

Aqueous micellar two-phase system composed of hyamine-type hydrophobically modified ethylene oxide and application for cytochrome P450 BM-3 separation

The hydrophobically modified ethylene oxide polymer, HM-EO, was modified with an alkyl halide to prepare a hyamine-type HM-EO, named N-Me-HM-EO, which could be used for forming N-Me-HM-EO/buffer aqueous micellar two-phase system. The critical micelle concentration of N-Me-HM-EO solution and the phase diagrams of N-Me-HM-EO/buffer systems were determined. By using this novel aqueous micellar two-phase system, the separation of cytochrome P450 BM-3 from cell extract was explored. The partitioning behavior of P450 BM-3 in N-Me-HM-EO/buffer systems was measured. The influences of some factors such as total proteins concentration, pH, temperature and salt concentration, on the partitioning coefficients of P450 BM-3 were investigated. Since the micellar aggregates in the N-Me-HM-EO enriched phase were positively charged, it was possible to conduct the proteins with different charges to top or bottom phases by adjusting pH and salt concentration in the system. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: the first extraction with N-Me-HM-EO/buffer system at pH 8.0, and the second extraction in the same system at pH 6.0. The recovery of P450 BM-3 was 73.3% with the purification factor of 2.5. The results indicated that the aqueous micellar two-phase system composed of hyamine modified polysoap has a promising application for selective separation of biomolecules depending on the enhanced electrostatic interactions between micelles and proteins.     

Liquid-liquid extraction of commercial and biosynthesized nisin by aqueous two-phase micellar systems

Nisin is a natural additive for conservation of food, and can also be used as a therapeutic agent. Nisin inhibits the outgrowth of spores, the growth of a variety of Gram-positive and Gram-negative bacteria. In this paper we present a potentially scalable and cost-effective way to purify commercial and biosynthesized in bioreactor nisin, including simultaneously removal of impurities and contaminants, increasing nisin activity. Aqueous two-phase micellar systems (ATPMS) are considered promising for bioseparation and purification purposes. Triton X-114 was chosen as the as phase-forming surfactant because it is relatively mild to proteins and it also forms two coexisting phases within a convenient temperature range. Nisin activity was determined by the agar diffusion assay utilizing Lactobacillus sake as a sensitive indicator microorganism. Results indicated that nisin partitions preferentially to the micelle rich-phase, despite the surfactant concentration tested, and its antimicrobial activity increases. The successful implementation of this peptide partitioning, from a suspension containing other compounds, represents an important step towards developing a separation method for nisin, and more generally, for other biomolecules of interest.     

Purification of a Bacteriocin-Like Inhibitory Substance Derived from Pediococcus acidilactici Kp10 by an Aqueous Micellar Two-Phase System

Aqueous micellar two-phase system (AMTPS) is an extractive technique of biomolecule, where it is based on the differential partitioning behavior of biomolecule between a micelle-rich and a micelle-poor phase. In this study, an AMTPS composed of a nonionic surfactant, Triton X-100 (TX-100) was used for purifying a bacteriocin-like inhibitory substance (BLIS) derived from Pediococcus acidilactici Kp10. The influences of the surfactant concentration and the effect of additives on the partitioning behavior and activity yield of the BLIS were investigated. The obtained coexistence curves showed that the mixtures of solutions composed of different surfactant concentrations (5–30% w/w) and 50% w/w crude load were able to separate into two phases at temperatures of above 60 °C. The optimum conditions for BLIS partitioning using the TX-100-based AMTPS were: TX-100 concentration of 22.5% w/w, CFCS load of 50% w/w, incubation time of 30 min at 75 °C, and back-extraction using acetone precipitation. This optimal partitioning resulted in an activity yield of 64.3% and a purification factor of 5.8. Moreover, the addition of several additives, such as sorbitol, KCl, dioctyl sulfosuccinate sodium salt, and Coomassie® Brilliant Blue, demonstrated no improvement in the BLIS separation, except for Amberlite® resin XAD-4, where the activity yield was improved to 70.3% but the purification factor was reduced to 2.3. Results from this study have demonstrated the potential and applicability of TX-100-based AMTPS as a primary recovery method for the BLIS from a complex fermentation broth of P. acidilactici Kp10. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2719, 2019     

Tuning micelles of a bioactive heptapeptide biosurfactant via extrinsically induced conformational transition of surfactin assembly

We have studied the effects of extrinsic environmental conditions on the conformation of surfactin, a heptapeptide biosurfactant from Bacillus subtilis, in aqueous solutions. It has been made clear that temperature, pH, Ca²⁺ ions and the synthetic nonionic surfactant hepta-ethylene glycol (C₁₂E₇) affect the conformation of surfactin in aqueous solutions. The β-sheet formation reached a maximum at 40°C both in presence and absence of (C₁₂E₇) and the nonionic surfactant enhances the β-sheet formation even at 25°C. Ca²⁺ induced the formation of a-helices and caused this transition at 0.3 mm with surfactin monomers or at 0.5 mm with surfactin micelles, but above these transition concentrations of Ca²⁺ β-sheets were observed. In micellar solution the β-sheet structure was stabilized at pH values below 7 or upon addition of Ca²⁺ in concentrations above 0.5 mm . Our results indicated that the bioactive conformation of surfactin is most likely the β-sheets when the molecules are assembled in micelles. The β-sheet structure in micelles could be retained by tuning the micelles. Surfactin micelles could be tuned in the bioactive conformation by manipulating pH, temperature, Ca²⁺ or (C₁₂E₇) concentrations in surfactin solutions. Our results strongly indicated that Ca²⁺ and other molecules (such as C₁₂E₇) may function as directing templates in the assembly and conformation of surfactin in micelles. Thus, we suggest environmental manipulation and template-aided micellation (TAM) as a new approach for preparing predesigned micelles, microemulsions or micro-spheres for specific application purposes. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Separating lysozyme from bacteriophage P22 in two-phase aqueous micellar systems

This communication demonstrates that two-phase aqueous mixed (nonionic/ionic) micellar systems have the potential for improving the separation of proteins from viruses. Specifically, two separation experiments were performed to show that the addition of the anionic surfactant sodium dodecyl sulfate (SDS) to the two-phase aqueous nonionic n-decyl tetra(ethylene oxide) (C(10)E(4)) micellar system increases the yield of a model net positively charged protein, lysozyme, in the micelle-rich phase from 75 to 95%, while still maintaining approximately the same yield of a model net negatively charged virus, bacteriophage P22, in the micelle-poor phase (97% vs. 98%).     

Hydrophobin (HFBI): A potential fusion partner for one-step purification of recombinant proteins from insect cells

Hydrophobins play an important role in binding and assembly of fungal surface structures as well as in medium-air interactions. These, hydrophobic properties provide interesting possibilities when purification of macromolecules is concerned. In aqueous micellar two-phase systems, based on surfactants, the water soluble hydrophobins are concentrated inside micellar structures and, thus, distributed to defined aqueous phases. This, one-step purification is attractive particularly when large-scale production of recombinant proteins is concerned. In the present study the hydrophobin HFBI of Trichoderma reesei was expressed as an N-terminal fusion with chicken avidin in baculovirus infected insect cells. The intracellular distribution of the recombinant fusion construct was analyzed by confocal microscopy and the protein subsequently purified from cytoplasmic extracts in an aqueous micellar two-phase system by using a non-ionic surfactant. The results show that hydrophobin and an avidin fusion thereof were efficiently expressed in insect cells and that these hydrophobic proteins could be efficiently purified from these cells in one-step by adopting an aqueous micellar two-phase system.     

Recovery of endo-polygalacturonase using polyethylene glycol-salt aqueous two-phase extraction with polymer recycling

The partitioning behaviour of endo-polygalacturonase (endo-PG) and total protein from a clarified Kluyveromyces marxianus fermentation broth in polyethylene glycol (PEG)-ammonium sulfate and PEG-potassium phosphate (pH=7) aqueous two-phase systems was experimentally investigated. Both the enzyme and total protein partitioned in the bottom phase for these two kinds of systems. The enzyme partitioning coefficient can be lower than 0.01 in PEG8000-(NH₄)₂SO₄ ATPS with a large phase volume ratio and a moderate tie-line length, which implies the possibility of concentration operation using aqueous two phase partitioning. An ion-exchange separation of high purification efficiency was applied to analyze the clarified and dialyzed fermentation broth. A total purification factor of only 2.3 was obtained, which indicated the high enzyme protein content in the total protein of the fermentation broth. Consequently, the main purpose for separating endo-PG is concentration rather than purification. A separation scheme using an aqueous two-phase extraction process with polymer recycling and a dialysis was proposed to recover endo-PG from the fermentation supernatant of K. marxianus for commercial purpose. A high enzyme recovery up to 95% and a concentration factor of 5 to 8 with a purification factor of about 1.25 were obtained using the single aqueous two-phase extraction process. More than 95% polymer recycled will not affect the enzyme recovery and purification factor. Dialysis was used mainly to remove salts in the bottom phase. The dialysis step has no enzyme loss and can further remove small bulk proteins. The total purification factor for the scheme is about 1.7.     

Pectinase partitioning in polyethylene glycol 1000/Na2SO4 aqueous two-phase system: statistical modeling of the experimental results

Statistical models concerning partitioning of pectinase in polyethylene glycol 1000/Na₂SO₄ aqueous two-phase system were established with response surface methodology. Concentrations of polyethylene glycol 1000 and Na₂SO₄ were selected as independent variables to evaluate their impact on parameters of partitioning in aqueous two-phase system—the partition coefficient of pectinase, purification factor and pectinase yield. An experimental space where over 2.5-fold purification was achieved, followed by over 90% yield of pectinase. The established models showed good prediction of partitioning parameters.     

On the meaning of enzymatic superactivity in reverse micelles

Summary Two types of superactivity can be defined. One, is with respect to the activity for a fixed average substrate concentration in the water pool; the pushing of the charged substrate by the likewise charged micellar surface is responsible for the superactivity and its bell-shaped dependence on the hydration ratio. The other, is with respect to the activity in a bulk aqueous solution having a substrate concentration equal to a fixed overall concentration [S^–]_ov in the entire reverse micellar solution. In this case, the pushing effect, the constraint of a fixed [S^–]_ov and the partitioning of the substrate in the surfactant layer are responsible for the bell-shaped dependence. Superactivity exists for low substrate partitioning in the surfactant layer, subactivity for high partitioning.     

5CN05 partitioning in an aqueous two-phase system: A new approach to the solubilization of hydrophobic drugs

Liquid–liquid extraction for the purification of molecules has been central to many advances in the pharmaceutical industry. These processes were developed based on the property that some polymer and/or micellar solutions present to separate into a concentrated phase and a diluted phase. Based on the differences in the physical and chemical environments of the two coexisting phases, and since both phases contain approximately 60–90% of water, liquid–liquid extraction provides a powerful alternative to both extract and solubilize a molecule. This paper examines the partition behavior of the synthetic drug, 2-[(3,4-dichlorine-benzylidene)-amino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile (5CN05), in an aqueous two-phase polymer system (ATPPS) and also in an aqueous two-phase micellar system (ATPMS). The results showed that both systems are favorable for extraction the 5CN05 drug high partition coefficient values (K_5CN05 > 200) and yield (Y_5CN05 > 99.48%) in the concentrated phase were achieved with the systems. However, the ATPPS generated a partition coefficient (K_5CN05) higher than the one obtained with ATPMS. The results suggest that both processes may be used for the extraction and concentration of molecules with hydrophobic characteristics, such as 5CN05. They also provide an optimal environment for the solubilization of such molecules, allowing for greater efficiency when purifying many classes of drugs.     

Structure of vesicles formed from non-ionic dialkylglycerol poly(oxyethylene) ether surfactants: effect of electrolyte and cholesterol

Five non-ionic dialkylglycerol poly(oxyethylene) ether surfactants, designated 2C_mE_n (where m, the number of carbons in each alkyl chain = 16 or 18, and n, the number of oxyethylene units = 12, 16 or 17) have been examined for their ability to form vesicles when dispersed in water or in an aqueous solution of 154 mM NaCl, alone or in the presence of 50 mol% cholesterol. Freeze fracture electron microscopy and light scattering showed that regardless of the hydrating fluid, all the non-ionic surfactants, with the exception of 2C₁₆E₁₇ and 2C₁₈E₁₇, formed vesicles in the absence of cholesterol – 2C₁₆E₁₇ and 2C₁₈E₁₇ instead formed micellar aggregates. All surfactants, however, formed vesicles in the presence of 50 mol% cholesterol. Small angle neutron scattering studies of the surfactant vesicles enabled the bilayer thickness and repeat distance (d-spacing) to be determined. The bilayers formed by all the non-ionic surfactants in the absence of cholesterol were surprisingly thin (∼50 Å for the E₁₂ containing surfactants and ∼64 Å for 2C₁₈E₁₆) most likely due to the intrusion of oxyethylene groups into the hydrophobic core of the bilayers. In contrast, however, the non-ionic surfactants exhibited a relatively large d-spacing of around ∼130–150 Å. The addition of 50 mol% cholesterol had a dramatic effect on the thickness of the vesicle bilayer, increasing its size by 10–20 Å, most probably because of an extrusion of oxyethylene from the hydrophobic region of the bilayer and/or a reduction in the tilt on the surfactant alkyl chains. Additionally the presence of cholesterol in a vesicle tended to reduce slightly both the d-spacing and the thickness of the water layer separating the bilayers. The presence of NaCl, even at the low concentrations used in the study, did affect the properties of the bilayer such that it reduced the d-spacing and, in the case of cholesterol-containing systems, also reduced bilayer thickness.     

Partitioning of Proteins using a Hydrophobically Modified Ethylene Oxide/SDS Aqueous Two-phase System

Summary A novel aqueous two-phase system containing hydrophobically modified ethylene oxide (HM-EO) and sodium dodecyl sulphate (SDS) was developed to enhance the selectivity of protein partitioning in two phases. Phase diagrams of HM-EO/H₂O and HM-EO/SDS/H₂O were measured, and the mechanism of interaction between HM-EO polymer and the anionic surfactant sodium dodecyl sulphate (SDS) was also discussed. It was found that the improvement of selectivity of protein partitioning was related to the increase of electrostatic potential difference between the two phases because of the charged network formed by mixed micelles of HM-EO and SDS in the bottom phase. With bovine serum albumin (BSA) and lysozyme as model proteins, some factors, such as pH, SDS concentration, conductivity and temperature of the system, were investigated for the influences of protein partition in HM-EO/SDS/H₂O systems. The results showed that the addition of SDS not only changed the phase behaviour, but also played an important role in protein partitioning.     

Molecular dynamics study of changes in physico-chemical properties of DMPC lipid bilayers by addition of nonionic surfactant C12E10

Changes in physico-chemical properties of dimyristoyl phosphatidylcholine (DMPC) lipid bilayers caused by the addition of 9.4 mol% nonionic surfactant decaoxyethylene monododecyl ethers (C₁₂E₁₀) have been investigated by molecular dynamics calculations. In spite of addition of single chain C₁₂E₁₀, the lipid bilayers showed an increase of membrane area. Isothermal area compressibility, which is a measure of membrane softness in lateral direction, also increased by 50% for DMPC/C₁₂E₁₀ mixed bilayers. Furthermore, the order parameter of C–H vector for DMPC acyl tails decreased. We found that these changes are caused by the hydrophilic head groups of C₁₂E₁₀ which are located near the glycerol backbone of the DMPC molecules and have bulky random coil conformation without any preferential ordered structures.     

Stimuli-responsive separation of proteins using immobilized liposome chromatography

The possibility of the stimuli-responsive separation of proteins was investigated using immobilized liposome chromatography (ILC) as novel aqueous two-phase systems. The specific capacity factor (k_s) of β-galactosidase, obtained by analysis of ILC, was varied by changing the pH of the solution and was maximized at the specific pH of 5 (k_s,max=5.57). The k_s values were found to correspond well with their local hydrophobicities, which can be determined by the aqueous two-phase partitioning method. The variation of k_s, therefore, indicates a change in the surface properties of a protein during conformational change under pH stimuli. A similar phenomenon is observed in the case of other proteins (α-glucosidase, k_s,max=11.3 at pH 4; carbonic anhydrase from bovine, k_s,max=6.53 at pH 4). The difference in the height and/or the position of the peaks of the k_s–pH curves of each protein suggests a difference in their pH denaturation in the ILC column. Based on these results, the mutual separation of the above proteins at pH 4 could be successfully performed by selecting their specific capacity factor as a design parameter.     

Liquid–liquid extraction of antimicrobial peptide P34 by aqueous two-phase and micellar systems

Antimicrobial peptide P34 is a promising biopreservative for utilization in the food industry. In this work, aqueous biphasic systems (ABS) and aqueous biphasic micellar systems (ABMS) were studied as prestep for purification of peptide P34. The ABS was prepared with polyethylene glycol (PEG) and inorganic salts and the ABMS with Triton X-114 was chosen as the phase-forming surfactant. Results indicate that peptide P34 partitions preferentially to PEG-rich phase and extraction with ammonium sulfate [(NH₄)₂SO₄], yielding a 75% recovery of the antimicrobial activity, specific activity of 1,530 antimicrobial units per mg of protein, and purification fold of 2.48. Protein partition coefficient and partition coefficient for the biological activity with (NH₄)₂SO₄ system were 0.48 and 64, respectively. Addition of sodium chloride did not affect recovery, but decreased protein amount in the PEG-rich phase, indicating a higher partition of biomolecules. ABMS did not yield good recovery of antimicrobial activity. Purification fold using PEG–(NH₄)₂SO₄ and 1.0?mol l^?1 sodium chloride was twice higher than that obtained by conventional protocol, indicating a successful utilization of ABS as a step for purification of peptide P34.