Refractive index matching to develop transparent polyaphrons: Characterization of immobilized proteins

Colloids and Surfaces B: Biointerfaces 142 (2016) 159–164

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Refractive index matching to develop transparent polyaphrons: Characterization of immobilized proteins Keeran Ward, David C. Stuckey ∗ Department of Chemical Engineering, Imperial College London, SW7 2AZ London, UK

a r t i c l e

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Article history: Received 6 October 2015 Received in revised form 11 February 2016 Accepted 16 February 2016 Available online 27 February 2016 Keywords: Protein Conformation Colloidal liquid aphrons (CLAs) Refractive index matching Adsorption Immobilization

a b s t r a c t Refractive index matching was used to create optically transparent polyaphrons to enable proteins adsorbed to the aphron surface to be characterized. Due to the significant light scattering created by polyaphrons, refractive index matching allowed for representative circular dichroism (CD) spectra and acceptable structural characterization. The method utilized n-hexane as the solvent phase, a mixture of glycerol and phosphate buffer (30% [w/v]) as the aqueous phase, and the non-ionic surfactants, Laureth4 and Kolliphor P-188. Deconvolution of CD spectra revealed that the immobilized protein adapted its native conformation, showing that the adsorbed protein interacted only with the bound water layer (“soapy shell”) of the aphron. Isothermal calorimetry further demonstrated that non-ionic surfactant interactions were virtually non-existent, even at the high concentrations used (5% [w/v]), proving that non-ionic surfactants can preserve protein conformation. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Characterization of protein structures at oil-water interfaces has been of interest for some years due to their inherent interfacial properties leading to their application in emulsion stability [1,2]. A variety of spectroscopic techniques have been used to assess conformational changes at the oil-water interface, such as Fourier transform infrared spectroscopy (FTIR) and tryptophan fluorescence spectroscopy, which have been used to investigate changes occurring on solid surfaces and stabilized emulsions [3,4], and these techniques have found that proteins undergo some conformational change at the oil-water interface. However, the quantification of protein secondary structure using FTIR is not a trivial process since it requires careful deconvolution (using curve fitting software), and solvent subtraction [5]. Circular dichroism (CD) has been used extensively to assess protein structure [6–8], and relies on the interaction between chromophores within the secondary structure of proteins and polarized light [9]. As this occurs, the chiral compounds of the protein backbone absorb light, allowing for excitation. Different structural contributions, such as helical and pleated sheets, relay different transitions during excitation leading to characteristic spectra [10].

∗ Corresponding author. E-mail addresses: [email protected] (K. Ward), [email protected], [email protected] (D.C. Stuckey). http://dx.doi.org/10.1016/j.colsurfb.2016.02.054 0927-7765/© 2016 Elsevier B.V. All rights reserved.

However, the use of CD to assess protein conformation at the oilwater interface has been difficult due to the high absorbance/light scattering of these systems, which hinders reliable CD spectra from being obtained. Studies by Husband and coworkers used glycerol and polyethylene glycol in the aqueous phase in an effort to create refractive index (RI) matched emulsions [11]. This modification created a fairly transparent emulsion, which was also capable of enabling high quality CD spectra to be obtained to determine protein conformation. Some of the conditions necessary for selecting an acceptable RI additive are as follows; a high RI, good water solubility, low absorbance over the CD range used, and be nonhalogenic, non-chiral and non-denaturing in nature. Although both glycerol and polyethylene glycol had all of the characteristics desirable for RI matching, the high levels of glycerol needed (58% (v/v)) can affect the conformation of proteins upon adsorption, and in solution [7,11]. Colloidal liquid aphrons (CLAs) differ from conventional emulsions in that their structural arrangement (micron sized oil droplets stabilized by surfactant bilayers) decreases the likelihood of phase separation. Also, unlike emulsions, CLAs do not need large volumes of additives for RI matching since only a small volume of aqueous phase is necessary for formulation. Past studies utilizing anionic surfactants has attributed surfactant interactions to superactivity as well as denaturation among CLA immobilized enzymes, with very little known about immobilized enzyme conformation [12–15]. The novelty in this research lies in the creation of RI matched polyaphrons that are very similar supports for pro-

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tein immobilization as conventional polyaphrons. Furthermore, the ability to characterize protein structure at these interfaces has not been attempted before in past research, and hence can provide valuable information about the interactions occurring during immobilization. In this study we present a novel method for formulating RI matched non-ionic polyaphrons for the characterization of four [4] (4) proteins, bovine serum albumin (BSA), ovalbumin, lysozyme and ␣-chymotrypsin, using CD. 2. Materials and methods 2.1. Materials The enzymes examined were; lysozyme from chicken egg white (EC 3.2.1.17, Mucopeptide N-acetylmuramylhydrolase, 90% pure, Sigma), Bovine Serum Albumin (BSA, 96% pure, Sigma Aldrich), lyophilized Albumin from chicken egg white (Ovalbumin, 98% pure, Sigma Aldrich,), and ␣-chymotrypsin from bovine pancreas (Sigma Aldrich, E.C. 3.4.21.1). Polyaphrons were manufactured using Mineral Oil (Sigma Aldrich), Tween 20 (Polyoxyethylene sorbitan monolaurate, 99% pure, Sigma Aldrich), Tween 80 (Polyoxyethylene sorbitan monooleate, 99% pure, Sigma Aldrich), Kolliphor P-188 (Poly (ethylene glycol)-block-poly (propylene glycol)-blockpoly(ethylene glycol), Sigma), Kolliphor-EL (Polyoxyl-35 castor oil, Sigma) and Triton X-100 (Polyethylene glycol tert-octylphenyl ether, TX-100, Sigma). n-Hexane (97% pure, VWR International), Laureth-4 (tetraethylene glycol monododecyl ether) and glycerol (99.5% pure, VWR International) were used for refractive index matching. Potassium phosphate monobasic (KH2 PO4, 99% Sigma) was used for buffering. The high-performance liquid chromatography grade organic solvent, acetonitrile (99.9%, VWR), and reagent grade trifluoroacetic acid (99.5%, Sigma) were used for assaying concentration. The water used throughout the experiment was distilled and deionised. 2.2. Instruments UV–vis spectrometry/UV–vis scanning spectrophotometer (UV1800 Shimadzu, UK) and High Performance Liquid Chromatography (HPLC Shimadzu, UK) were used for analyzing enzyme samples using a silica-based C4HPLC column (Phenomenex, UK). An overhead stirrer (Heidolph RZR 2020) was used for polyaphron preparation and a vortex (Fisher Scientific) used for mixing, while a Biofuge Stratos centrifuge (Heraeus Instruments) was used for CLA separation. Disposable syringes (B.Braun Melsungen AG) and 0.22 ␮m syringe filters (Millipore Co.) were used to remove particulates prior to sample analysis. Circular dichroism (CD) spectral measurements were performed on a Jasco J715 Spectropolarimeter using a 1-mm quartz cell, while Isothermal calorimetry was performed using a Micro-CAL titration calorimeter (Micro Cal Inc., USA). Particle Size measurements were performed using the Mastersizer 2000 (Malvern instruments). 2.3. Protein preparation BSA, ovalbumin and lysozyme protein samples were prepared in 20 mM potassium phosphate (monobasic) buffer, while ␣chymotrypsin samples were made up via stock solutions using 1 mM HCL solution consisting of 2 mM CaCl2 , and further diluted to 0.08 mM. CaCl2 was necessary for protease stability.

stirred foaming aqueous phase (1% w/v Tween 20 in lysozyme solution) using overhead stirring until the required phase volume ratio (PVR = Vorg × Vaq −1 ) was reached. The solvent was added at a flow rate of 0.3 mL min−1 at a stirrer speed of 700 rpm; after addition of the total volume of oil, the formulation was sheared using the same apparatus at 1000 rpm in order to achieve the desired droplet size (∼12 ␮m). The resulting formulation was very viscous with a creamy white appearance, and showed no phase separation over a period of several weeks. Polyaphron formulations were made up to a phase volume ratio (PVR) of 8 and diluted to PVR 4. PVR 8 was the optimum choice for the PVR taking into consideration viscosity limitations as well as uniform particle sizes. However, PVR 8 formulations became difficult to measure volumes in, and hence the formulation was consequently diluted for easier estimations, while the droplet size and distribution was unaffected [16]. 2.5. Refractive index matched polyaphrons (RIMPs) The aqueous phase consisted of 5% (w/v) Kolliphor P-188, 25% (w/v) glycerol, and 70% (w/v) phosphate buffer. Protein concentrations investigated were 2–3 mg mL−1 , while the solvent phase consisted of 1% (w/v) Laureth-4 in n-hexane. Approximately 12 mL of the solvent phase was carefully added to 3 mL of the aqueous phase using a pipette (at a rate of 0.5 mL min−1 ), at a stirrer speed of 260 rpm. Upon addition of all the solvent phase, 4–5 drops of glycerol were then added to create a completely transparent polyaphron formulation. A large volume of glycerol (∼30% w/v) was needed to correct for the changes in RI due to the uptake of water within the “soapy shell” of the aphrons. Blank RIMPs (without immobilized protein), as well as samples of the aqueous phase, were used as controls in the CD experiments. 2.6. CLA manufacture, sample preparation and protein assay Polyaphron samples were dispersed into a bulk continuous buffer phase at a ratio of the dispersed phase to the bulk of approximately 40% CLA: 60% water. Samples were vortex mixed for 1–2 min for homogeneity and allowed to settle for 1 h; after settling two distinct layers were observed, and the samples were centrifuged at 8500g for 25 min for further separation without CLA deterioration. A 1–2 mL sample of the supernatant was pipetted and filtered using 0.22 ␮m syringe filters to ensure a CLA free sample for protein assaying. Lysozyme samples (100 ␮L) were injected into a Phenomenex C4HPLC column equilibrated with 0.1% trifluoroacetic acid (TFA) in water (mobile phase A). The column was eluted using 0.1% TFA in acetonitrile (mobile phase B) using a gradient from 2 to 30% B in 15 min, and then a ramp from 30 to 90% for another 15 min at a flow rate of 1.5 mL min−1 . Retention time (tR ) for lysozyme was around 13.8 min. Chromatograms were analyzed using Origin Pro 9 software. 2.7. Error analysis Experimental errors for all results were measured by calculating the coefficient of variance: ␴ COV = (1)  where ␴ is the standard deviation, and ␮ is the mean value. Samples (n = 4) were reproduced using 4 replicates giving a COV of <±10%. 2.8. Particle size analysis

2.4. Polyaphron formulation Polyaphron phases were made by the dropwise addition of mineral oil/surfactant solution (1% w/v Tween 80) from a burette into a

A Malvern Particle Size analyzer (Mastersizer 2000) was used to quantify the radius of the CLA. Polyaphron samples were diluted by adding 0.1 mL of the formulation directly into the dispersion

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unit (120 mL volume) filled with deionised water, and stirred at 2400 rpm. For each sample 6 readings were taken, with the average particle size quoted as D [4,3], giving a COV of ±6%. 2.9. Isothermal calorimetry (ITC) Measurements were performed using a VP-ITC (Micro Cal) titration calorimeter, and the method was adapted from the work by Al-Anber (2013) [26]. The unit consisted of a reference and sample cell, roughly 1.4 mL in volume, and was insulated by an adiabatic shield allowing for careful feedback to maintain both cells at the same temperature. The sample cell and syringe were washed with distilled water before each run, and then the sample cell was loaded with lysozyme solution, while the syringe was loaded with surfactant. The initial delay was 60 s, reference power 10 ␮cal/s, and the filter was 2 s; the ITC unit was calibrated at 25 ◦ C. A total of 17–20 injections were run for each sample, with an injection time of 20 s, and a time interval between injections of 210 s. The solution in the sample cell was stirred at 315 rpm using a rotating paddle, and the heat of dilution for both the protein and surfactant was measured and subtracted to obtain the heat of adsorption. Samples were run in triplicate with a COV <±5%. 2.10. Circular dichroism (CD) The CD spectra of native and immobilized proteins were recorded over the far-UV wavelength range of 200–240 nm with a scan at 20 ◦ C in a thermostatic cell holder. The path length was 1 mm, the step resolution 0.5 nm and the bandwidth 1 nm; the scan speed was 10 nm min−1 . 1 mL of the RIMP formulation was pipetted into a syringe fitted with a needle, and then the sample was added to the CD cuvette; sample cuvettes were then centrifuged at 2000g for 5 min to expel air bubbles. Dissolved protein samples consisted of native protein solutions formulated using the same aqueous phase as that used for the RIMP formulation. Samples of the aqueous phase as well as blank RIMPs were measured and subtracted from both the immobilized and native protein spectra, with data presented as ellipticities (␪ millidegree). The observed ellipticities were converted into molar ellipticities based on the mean molecular mass per residue of each protein. Average spectra of the replicate scans were analysed using deconvolution software (CDNN program version 2.1), which calculates the secondary structure of the peptide by comparison with a base set of 13 known protein structures. The COV was <±5%. 3. Results and discussion 3.1. Surfactant—lysozyme interactions and CLA-lysozyme adsorption In an effort to understand the effects of non-ionic surfactants on adsorbed protein conformation, a study into the adsorption of lysozyme using different non-ionic surfactants was carried out. An estimate of the amount of protein adsorbed was carried out using a mass balance, as illustrated in Eq. (1): Mi = CL VL − CS VS

(1)

where, Mi is the mass immobilized [mg], CL is the enzyme loading (concentration) examined [mg mL−1 ], VL is the volume of enzyme solution used [mL], CS is the enzyme concentration in the supernatant [mg ml−1 ], and vs is the volume of the supernatant [mL]. The amount immobilized was calculated as the mass immobilized per CLA surface, as described in Eq. (2) [17]: Mass Adsorbed per CLA Surface = Mr (2)where, r is the radius of 3V the CLA [m], and V is the volume of oil used for manufacture [m3 ].

Fig. 1. Lysozyme adsorption as a function of changing non-ionic surfactant. Enzyme concentration 4 mg mL−1 at pH 6.2. Error bars report SD, n = 4.

Percentage immobilization was approximated by comparing the immobilized mass to the initial mass of protein used: Percent Immobilization (w/w) =

Mi M

(3)

where, M is the mass of protein initially used during formulation. The bar chart (Fig. 1) shows that lysozyme immobilization was relatively unchanged using different surfactants; the maximum amount of lysozyme adsorbed was 0.27 mg.m2 , accounting for 27% (w/w) immobilization (Eq. (3)). Several studies have been carried out using non-ionic surfactants for protein solublization and extraction due to their non-denautring nature [18–20]. Furthermore, several reports have indicated high activity retention amongst enzymes interacting with non-ionic reverse micelles, showing that proteins can maintain activity at concentrations at or higher than the critical micelle concentration [21,22]. Some studies suggest that specific groups of non-ionic surfactants, in particular Tweens and Sorbitans, can possess a negative charge able to induce electrostatic interactions [18,23]. Based on the results in Fig. 1, there is no change in the adsorption profile of lysoyzme (postively charged, pI = 11) onto non-ionic CLAs, and hence surfactant interactions are most likely too weak to support immobilization. For non-ionic CLAs, the main forces contributing to adsorption are hydrophobic interactions due to surfactant interactions, as well as those of the non-polar solvent phase [15,24]. Due to the charge interactions of lysozyme, it is possible that repulsion between protein molecules could decrease immobilization, however, for the adsorption of proteins onto hydrophobic interfaces, hydrophobic interactions can overcome electrostatic interactions, allowing for immobilization [25]. Fig. 2 presents the calorimetric traces for lysozyme and surfactants Triton X-100 (TX-100) and Kolliphor P-188. These results show that the heat generated from surfactant dilution was much greater than that from lysozyme-surfactant interactions, and changing the concentration of lysozyme and surfactant by a factor of 10 had no influence on the interaction. The integral enthalpy change for an interaction using ITC was recorded by estimating the difference between the heat of dilution and that of the interaction between lysozyme and surfactant molecules. However, since the heat of dilution was almost identical to that of the interaction, the enthalpy change was recorded as null. The results of this experiment differ considerably from those researchers investigating the interaction of lysozyme with cationic surfactants where the

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Fig. 2. ITC isotherms of (a) Kolliphor P-188 dilution, (b) TX-100 dilution, (c) Lysozyme- Kolliphor P-188 and, (d) Lysozyme-TX-100. Enzyme concentration 10 mg mL-1 , surfactant concentration 5% (w/v) at pH 6.2.

enthalpy changes were much larger than those of surfactant dilution [26]. Hence, these results suggest that the interaction between lysozyme and non-ionic surfactants was relatively small. By comparing the results from Figs. 1 and 2, it is clear that hydrophobic interactions due to the presence of a non-polar solvent drives lysozyme adsorption rather than surfactant interactions.

3.2. Immobilized protein characterization Fig. 3 shows the spectra for both immobilized and dissolved proteins; each individual spectrum has unique minimum points which indicate distinctive structural properties. With ␣-chymotrypsin, a negative shoulder is observed at 230 nm corresponding to the histidine-40-tryptophan-141 complex [27]. For BSA, two negative peaks at 209 and 222 nm indicate the transitions of amide bonds within the helical conformation of the protein [28]. Lysozyme gives similar peak contributions, while ovalbumin has a major peak around 222 nm. For the immobilized enzyme, each spectrum shows the same unique characteristics of its freely dissolved counterparts. Comparing the results in Table 1 shows that the conformation of the immobilized protein is maintained upon adsorption to the CLA surface. The spectra and deconvolution data is consistent with the findings of Banerjee and Pal [27] for ␣-chymotrypsin, Huntington and coworkers [29] for ovalbumin, Reed and coworkers [30] for BSA, and Greenfield [9] for lysozyme. Protein adsorption

Table 1 Deconvoluted CD spectra for Dissolved and Immobilized Proteins. Protein

Deconvoluted data (%) ␣-Helix

␤-Sheet

␤-Turn

Random coil

Immobilized protein ␣-Chymotrypsin BSA Ovalbumin Lysozyme

9 53 28 30

34 10 21 19

22 14 18 25

35 23 33 26

Dissolved protein ␣-Chymotrypsin BSA Ovalbumin Lysozyme

10 54 29 32

32 9 22 18

23 14 17 23

35 23 32 27

at oil/water interfaces almost always results in conformational changes due to unfolding, as non-polar residues become available for interaction; the adsorption kinetics for proteins at an interface occurs in stages. Firstly, the protein is transported to the surface from the bulk phase, allowing for surface interactions to occur. Secondly, spreading occurs due to the extent of the interactions, whether hydrophobic or electrostatic. The spreading rate depends both on the conformational changes arising from interactions, as well as protein-protein interactions due to the proximity of neighbouring molecules [31,32].

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Fig. 3. Far-UV circular dichroism spectra of immobilized and dissolved ␣-chymotrypsin (a), BSA (b), cysozyme (c), and ovalbumin (d).

The effect of both hydrophobic and electrostatic interactions on conformation depends on the nature and charge of the protein. For electrostatic interactions, the charge of the protein and the surface induces binding which allows for adsorption. However, as the adsorption rate increases, repulsive forces arising from like charges can limit the amount of protein adsorbed [33]. For hydrophobic surfaces, adsorption occurs regardless of the nature of electrostatic forces since the driving force for adsorption is determined by the hydrophobic exterior of the protein [34]. In most cases, hydrophobic surfaces lead to a greater adsorbed layer due to a large entropy gain arising from the interaction with the surface; as this occurs, the likelihood for unfolding increases [33]. Lysozyme has been known to lose small amounts of ␣-helix upon adsorption to interfaces [35]. However, for proteins such as BSA, ␤-casein and myoglobin, drastic reductions in secondary structure have been observed [7,11,28]. The nature of the solvent phase has a direct effect on the level of conformational change induced upon adsorption [36,37], and interactions with highly nonpolar solvents results in dehydration and a consequent increase in unfolding. For the RIMPs generated with hexane, adsorption to the solvent core directly results in conformational changes, and hence the results suggest that immobilization occurs within the aqueous shell of the polyaphron. The structure of aphrons has been debated in the literature over the past decades. Sebba [17] proposed that polyaphrons are comprised of an inner solvent core encapsulated by a surfactant bilayer. Studies by Princen [38] showed that these

systems are similar to high internal phase ratio emulsions (HIPREs), being both polyhedral in structure, but differing in the presence of a second surfactant. The presence of this polyhedral structure is the direct result of the closed packing arrangement of the aphrons. According to Sebba [17], the presence of a thin aqueous film or “soapy shell” was directly responsible for the inherent stability of these systems. Lye and Stuckey [39] investigated the structure of aphron dispersions and found that these systems do in fact possess a soapy shell, with a thickness close to what was first postulated by Sebba. The results suggest that upon adsorption, protein conformation is preserved due to the hydration effect induced by the “soapy shell” of the aphron. Studies have also shown that in the presence of small amounts of glycerol [35% (v/v)], proteins exhibit very little change in conformation [40]. This is due to the preferential hydration of glycerol since it does not significantly disrupt the hydrophobic interactions necessary for a stable conformation [41]. The results also suggest that non-ionic surfactant-interactions do not induce large conformational changes in the immobilized protein; this was evident by comparing ITC data in Fig. 2 with that of CD data in Table 1. Nonionic surfactants are known to preserve protein conformation since they do not cooperatively bind to protein molecules [42]. The presence of a water layer within the CLA structure is similar to the water pool in reverse micelle systems. In many cases, protein conformation has been preserved due to a hydration shell maintained by the water pool [43]. A study by Yan and coworkers [44] have

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revealed that the microstructure of CLAs possess reverse micelles (microemulsions) in both the solvent and aqueous phases. It is possible that the presence of these micelles can contribute to a level of hydration necessary to preserve conformation upon adsorption to the CLA surface. 4. Conclusions In this study we present a novel method of formulating refractive index matched polyaphrons to enable us to characterise immobilized proteins. The results showed that protein adsorption was mainly attributed to hydrophobic interactions resulting from the non-polar solvent core of the aphron. Furthermore, CD spectra suggested that proteins mainly interacted with the “soapy shell” of the polyaphron allowing the protein to adopt a hydrated conformation, with very few changes in structure being observed between the immobilized and dissolved states. The hydration required could be attributed to both the bound water of the “soapy shell”, as well as microemulsions present in the aqueous phase of the polyaphron. Finally, insight into surfactant interactions indicates that non-ionic surfactants do not bind to proteins, even at high concentrations, allowing for the preservation of protein conformation upon immobilization. Acknowledgments The authors are grateful for the partial funding received from MC2 Biotek, Derek Wheeler, Stephen Lenon and Fraser Steele from Drug Delivery Solutions for their assistance in developing the methodology, and Hanna Barriga for assisting in carrying out ITC experiments. References [1] D.G. Dalgleish, Food emulsions—their structures and structure-forming properties, Food Hydrocoll. 20 (4) (2006) 415–422. [2] P.J. Wilde, Interfaces: their role in foam and emulsion behaviour, Curr. Opin. Colloid Interface Sci. 5 (3–4) (2000) 176–181. [3] Y. Fang, D.G. Dalgleish, Conformation of beta-lactoglobulin studied by FTIR: effect of pH, temperature, and adsorption to the oil-water interface, J. Colloid Interface Sci. 196 (2) (1997) 292–298. [4] V. Rampon, C. Genot, A. Riaublanc, A. Anton, M.A.V. Axelos, D.J. McClements, Front-face fluorescence spectroscopy study of globular proteins in emulsions: influence of droplet flocculation, J. Agric. Food Chem. 51 (9) (2003) 2490–2495. [5] X.L. Qi, C. Holt, D. McNulty, D.T. Clarke, S. Brownlow, G.R. Jones, Effect of temperature on the secondary structure of beta-lactoglobulin at pH 6.7, as determined by CD and IR spectroscopy: a test of the molten globule hypothesis, Biochem. J. 324 (1997) 341–346. [6] T. Yamamoto, N. Fukui, A. Hori, Y. Matsui, Circular dichroism and fluorescence spectroscopy studies of the effect of cyclodextrins on the thermal stability of chicken egg white lysozyme in aqueous solution, J. Mol. Struct. 782 (1) (2006) 60–66. [7] B.T. Wong, J.L. Zhai, S.V. Hoffmann, M.I. Aguilar, M. Augustin, T.J. Wooster, et al., Conformational changes to deamidated wheat gliadins and beta-casein upon adsorption to oil-water emulsion interfaces, Food Hydrocoll. 27 (1) (2012) 91–101. [8] P.W.J.R. Caessens, W.F. Daamen, H. Gruppen, S. Visser, A.G.J. Voragen, beta-lactoglobulin hydrolysis. 2. Peptide identification, SH/SS exchange, and functional properties of hydrolysate fractions formed by the action of plasmin, J. Agric. Food Chem. 47 (8) (1999) 2980–2990. [9] N. Greenfield, Methods to estimate the conformation of proteins and polypeptides from circular dichroism data, Anal. Biochem. 235 (1) (1996) 1. [10] N. Sreerama, R.W. Woody, Computation and analysis of protein circular dichroism spectra, Methods Enzymol. 383 (2004) 318–351. [11] F.A. Husband, M.J. Garrood, A.R. Mackie, G.R. Burnett, P.J. Wilde, Adsorbed protein secondary and tertiary structures by circular dichroism and infrared spectroscopy with refractive index matched emulsions, J. Agric. Food Chem. 49 (2) (2001) 859–866. [12] G.J. Lye, Stereoselective Hydrolysis of dl-Phenylalanine methyl ester and Separation of l-Phenylalanine using aphron immobilised alpha-chymotrypsin, Biotechnol. Tech. 11 (8) (1997) 611–616. [13] G.J. Lye, M. Rosjidi, O.P. Pavlou, D.C. Stuckey, Immobilization of Candida cylindracea lipase on colloidal liquid aphrons (CLAs) and development of a continuous CLA-membrane reactor, Biotechnol. Bioeng. 51 (1) (1996) 69.

[14] S.B. Lamb, D.C. Lamb, S.L. Kelly, D.C. Stuckey, Cytochrome P450 immobilisation as a route to bioremediation/biocatalysis, FEBS Lett. 431 (3) (1998) 343. [15] S.B. Lamb, D.C. Stuckey, Enzyme immobilisation on colloidal liquid aphrons (CLAs): the influence of protein properties, Enzyme Microb. Technol. 24 (8–9) (1999) 541. [16] K. Matsushita, A.H. Mollah, D.C. Stuckey, C. Delcerro, A.I. Bailey, Predispersed solvent-extraction of dilute products using colloidal gas aphrons and colloidal liquid aphrons—aphron preparation, stability and size, Colloids Surf. 69 (1) (1992) 65. [17] F. Sebba, Foams and Biliquid Foams-Aphrons, John Wiley & Sons Ltd., 1987. [18] M. Vasudevan, J.M. Wiencek, Mechanism of the extraction of proteins into Tween 85 nonionic microemulsions, Ind. Eng. Chem. Res. 35 (4) (1996) 1085–1089. [19] K. Naoe, O. Ura, M. Hattori, M. Kawagoe, M. Imai, Protein extraction using non-ionic reverse micelles of Span 60, Biochem. Eng. J. 2 (2) (1998) 113–119. [20] Y.J. Nikas, C.L. Liu, T. Srivastava, N.L. Abbott, D. Blankschtein, Protein partitioning in 2-phase aqueous nonionic micellar solutions, Macromolecules 25 (18) (1992) 4797–4806. [21] G.A. Ayala, S. Kamat, E.J. Beckman, A.J. Russell, Protein extraction and activity in reverse micelles of a nonionic detergent, Biotechnol. Bioeng. 39 (8) (1992) 806–814. [22] Y. Yamada, R. Kuboi, I. Komasawa, Extraction of enzymes and their activities in aot reverse micellar systems modified with nonionic surfactants, J. Chem. Eng. Jpn. 27 (3) (1994) 404–409. [23] M. Vasudevan, K. Tahan, J.M. Wiencek, Surfactant structure effects in protein separations using nonionic microemulsions, Biotechnol. Bioeng. 46 (2) (1995) 99–108. [24] S.B. Lamb, D.C. Stuckey, Enzyme immobilization on colloidal liquid aphrons (CLAs): the influence of system parameters on activity, Enzyme Microb. Technol. 26 (8) (2000) 574. [25] S.B. Lamb, Enzyme Immobilization on Colloidal Liquid Aphrons (CLAs) and the Development of a Continuous Membrane Bioreactor [PhD], Imperial College London, 1999. [26] Z.A. Al-Anber, Thermodynamic studies of interaction between cetylpyridinium bromide and lysozyme by means of calorimetric titrations, J. Therm. Anal. Calorim. 111 (1) (2013) 823–830. [27] D. Banerjee, S.K. Pal, Conformational dynamics at the active site of alpha-chymotrypsin and enzymatic activity, Langmuir 24 (15) (2008) 8163–8168. [28] L. Day, J.L. Zhai, M. Xu, N.C. Jones, S.V. Hoffmann, T.J. Wooster, Conformational changes of globular proteins adsorbed at oil-in-water emulsion interfaces examined by synchrotron radiation circular dichroism, Food Hydrocoll. 34 (1) (2014) 78–87. [29] J.A. Huntington, P.A. Patston, Gettins PGW S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-Inserted serpins, Protein Sci. 4 (4) (1995) 613–621. [30] R.G. Reed, R.C. Feldhoff, O.L. Clute, T. Peters, Fragments of bovine serum-albumin produced by limited proteolysis—conformation and ligand-binding, Biochemistry (NY) 14 (21) (1975) 4578–4583. [31] M. Van der Veen, M.C. Stuart, W. Norde, Spreading of proteins and its effect on adsorption and desorption kinetics, Colloid Surf. B 54 (2) (2007) 136–142. [32] W. Norde, C.E. Giacomelli, Conformational changes in proteins at interfaces: from solution to the interface, and back, Macromol. Symp. 145 (1999) 125–136. [33] M. Kleijn, W. Norde, The adsorption of proteins from aqueous solution on solid surfaces, Heterogen. Chem. Rev. 2 (3) (1995) 157–172. [34] K. Nakanishi, T. Sakiyama, K. Imamura, On the adsorption of proteins on solid surfaces, a common but very complicated phenomenon, J. Biosci. Bioeng. 91 (3) (2001) 233–244. [35] W. Norde, J.P. Favier, Structure of adsorbed and desorbed proteins, Colloids Surf. 64 (1) (1992) 87–93. [36] A. Zaks, A.M. Klibanov, The effect of water on enzyme action in organic media, J. Biol. Chem. 263 (17) (1988) 8017–8021. [37] J.A. Rupley, E. Gratton, G. Careri, Water and globular proteins, Trends Biochem. Sci. 8 (1) (1983) 18. [38] H.M. Princen, Pressure volume surface-area relationships in foams and highly concentrated emulsions—role of volume fraction, Langmuir 4 (1) (1988) 164–169. [39] G.J. Lye, D.C. Stuckey, Structure and stability of colloidal liquid aphrons, Colloid Surf. A 131 (1–3) (1998) 119. [40] K. Gekko, S.N. Timasheff, Mechanism of protein stabilization by glycerol— preferential hydration in glycerol-water mixtures, Biochemistry (NY) 20 (16) (1981) 4667–4676. [41] Y.L. Khmelnitsky, Engineering biocatalytic systems in organic media with low water content, Enzyme Microb. Technol. 10 (12) (1988) 710. [42] Z. Wasylewski, A. Kozik, Protein – nonionic detergent interaction – interaction of bovine serum-albumin with alkyl glucosides studied by equilibrium dialysis and infrared spectroscopy, Eur. J. Biochem. 95 (1) (1979) 121. [43] N.L. Klyachko, N.G. Bogdanova, A.V. Levashov, K. Martinek, Micellar enzymology —superactivity of enzymes in reversed micelles of surfactants solvated by water organic cosolvent mixtures, Collection Czech. Chem. C 57 (3) (1992) 625–640. [44] Y.L. Yan, N.S. Zhang, C.T. Qu, L. Liu, Microstructure of colloidal liquid aphrons (CLAs) by freeze fracture transmission electron microscopy (FF-TEM), Colloid Surf. A 264 (1–3) (2005) 139–146.