Lysozyme extraction from egg white using reverse micelles in a Graesser contactor: mass transfer characterization

The gentle mixing characteristics of a Graesser contactor can help to avoid the formation of stable emulsions, which is one advantage of this type of contactor when used with reversed micellar extraction. In this study, the performance of the Graesser contactor in lysozyme extraction from hen egg white is investigated. The concentration profile of lysozyme in the aqueous and organic phases indicated that, while substantial axial mixing occurred in the contactor, the extraction yield was in the range of 97% to 99%. The number of mass transfer units (N(ox)) was determined using a diffusion model, and the influence of aqueous-to-organic phase flow ratio, rotor speed, and total throughput on contactor performance was studied. It was found that the diffusion model could describe quite well the extraction of lysozyme from hen egg white using reversed micelles. The optimal conditions for the extraction at steady state were found to be a rotor speed of 5 rpm, an aqueous-to-organic phase flow ratio of 60:20 mL/min, and a total throughput of 80 mL/min. In addition, back-extraction was also performed using the conventional method (1.5 M KBr at pH 11.5) in the contactor. It was found that this mass transfer was not well described by a diffusion model, although 85% of the lysozyme could be recovered with the operating conditions used: a rotor speed of 10 rpm, and an aqueous-to-organic flow rate of 10:10 mL/min.     

Affinity extraction of proteins with a reversed micellar system composed of Cibacron Blue-modified lecithin

Crude soybean lecithin was used as a novel surfactant to form reversed micelles in n-hexane. Cibacron Blue F-3GA (CB) was directly immobilized to the reversed micelles by a two-phase reaction. The reversed micellar system without CB showed low solubilizing capacity for low molecular weight proteins, lysozyme, and cytochrome c due to the weak electrostatic interactions. The introduction of CB significantly increased the solubilization of lysozyme because of its affinity binding to CB but showed no effect on the solubilization of cytochrome c since it did not bind to CB. Although bovine serum albumin had an affinity for CB, it was not extracted to the reversed micelles containing CB because its high molecular weight resulted in a significant steric hindrance effect. Thus the reversed micellar system had a high selectivity resulting from both biospecific and steric hindrance effects. The extraction yield of lysozyme decreased significantly with increasing ionic strength. Therefore, the back extraction of lysozyme was carried out using a stripping solution with an ionic strength of 0.865 mol/L. The overall recovery yield of lysozyme after back extraction could be increased to 87% by stripping for 2 h. The recovered lysozyme exhibited an activity equivalent to native lysozyme, and its secondary structure was also unchanged.     

Protein refolding mediated by reverse micelles of Cibacron Blue F-3GA modified nonionic surfactant

An affinity-based reverse micellar system formulated with nonionic surfactant was applied to the refolding of denatured-reduced lysozyme. The nonionic surfactant of sorbitan trioleate (Span 85) was modified with Cibacron Blue F-3GA (CB) as an affinity surfactant (CB-Span 85) to form affinity-based reverse micelles in n-hexane. The water content of 15 was found optimal for lysozyme refolding in the reverse micellar system of 62.7 mmol/L Span 85 with coupled CB of 0.3 and 0.5 mmol/L. In addition, the operating conditions such as pH and the concentrations of urea and redox reagents were optimized. Under the optimized conditions, complete renaturation of lysozyme at 3-3.5 mg/mL was achieved, whereas dilution refolding in the bulk aqueous phase under the same conditions gave much lower activity recovery. Moreover, the secondary structure of the refolded lysozyme was found to be the same as the native lysozyme. Over 95% of the refolded lysozyme was recovered from CB-Span 85 reverse micelles by a stripping solution of 0.5 mol/L MgCl(2). Thus, the present system is advantageous over the conventional reverse micellar system formed with ionic surfactants in the ease of protein recovery.     

Lysozyme extraction from hen egg white in an aqueous two-phase system composed of ethylene oxide–propylene oxide thermoseparating copolymer and potassium phosphate

The recovery and purification of lysozyme from hen egg white has been investigated in an aqueous two-phase systems composed of thermoseparating random copolymers of ethylene oxide (EO), propylene oxide (PO) and potassium phosphate. In the primary extraction step lysozyme was satisfactorily partitioned to the top polymer-rich phase in a system composed of 40% (w/w) EO₅₀PO₅₀, 10% (w/w) potassium phosphate, and 0.85 M sodium chloride at pH 9.0, diluted 3-fold with crude egg white, where contaminating proteins were discarded in the bottom phosphate-rich phase. After the primary phase separation the upper EO₅₀PO₅₀ phase was removed and subjected to temperature-induced (65 °C) phase separation, which resulted in the partitioning of pure lysozyme to the top water phase. The separation system was found to be efficient in achieving the purification of lysozyme in a high yield of 85% and specific activity of 32,300 U/mg of protein, with a purification factor of 16.9 and a concentration of lysozyme in the water phase of 2.3 g/l in two extraction steps.     

Effect of hexanol as a cosolvent on partitioning and mass transfer rate of protein extraction using reversed micelles of CB-modified lecithin

Using the affinity-based reversed micelles composed of Cibacron Blue F3G-A (CB) modified soybean lecithin, the effect of hexanol as a cosolvent on the extraction of lysozyme and bovine serum albumin (BSA) was investigated. The water concentration in the reversed micelles increased significantly with increasing hexanol concentration. The partition coefficient of lysozyme could be increased by over 12-fold by introducing hexanol of higher than 0.5 vol%. However, the transfer of BSA was hardly affected because its high molecular weight resulted in a strong steric hindrance effect. The enzymatic activity of lysozyme was nearly 100% retained after undergoing the extraction process with the CB–lecithin micelles containing 3 vol% hexanol. The partitioning isotherms of lysozyme in the CB–lecithin micelles with and without hexanol addition were expressed by the Langmuir equation. The partitioning capacity of lysozyme was nearly increased twofold by introducing 3 vol% hexanol to the CB–lecithin micelles and reached 2.12 g/l. The cosolvent hexanol revealed insignificant effect on the mass transfer rate, and in both the systems with and without hexanol, the mass transfer rate in back extraction was 5–10 times slower than that in the forward extraction. This phenomenon was similar to that in conventionally employed ionic surfactant systems. The result suggests that in the present affinity-based reversed micelles, the interfacial resistance also played a more important role in back extraction than in forward extraction.     

Purification of industrial alphaamylase by reversed micellar extraction

The purification of industrial alpha-amylase by liquid-liquid extraction with Aliquat 336 reversed micellar solution as the extractant was studied. Seven kinds of Aliquat 336 reversed micellar solution, formed by using seven kinds of straight chain alkyl alcohols as cosolvent, have been utilized to extract industrial a-amylase. It was found that these seven kinds of reversed micellar solution can all achieve a high protein transfer efficiency in the forward extraction process. After a full forward and backward extraction cycle, however, only the reversed micelles with n-butanol as the cosolvent was found to be able to maintain the activity of alpha-amylase in the stripping solution. By using the reversed micelles of Aliquat 336/isooctane/1% (v/v) n-butanol to perform a full extraction cycle, it was found that 85% of the total activity of alpha-amylase in the industrial a-amylase could be recovered at the end of an extraction cycle and the specific activity of alpha-amylase could be concentrated about 1.5-fold; meanwhile, most of the neutral protease in the industrial a-amylase could be removed. The separation factor of alpha-amylase to neutral protease at the end of an extraction cycle can reach about 10. (c) 1995 John Wiley & Sons, Inc.     

Thermoprecipitation of lysozyme from egg white using copolymers of N-isopropylacrylamide and acidic monomers

Thermoprecipitation of lysozyme from egg white was demonstrated using copolymers of N-isopropylacrylamide with acrylic acid, methacrylic acid, 2-acryloylamido-2-methylpropane-sulfonic acid and itaconic acid, respectively. Polymers synthesized using molar feed ratio of N-isopropylacrylamide:acidic monomers of 98:2 exhibited lower critical solution temperatures in the range of 33--35 degrees C. These polymers exhibited electrostatic interactions with lysozyme and inhibited its bacteriolytic activity. The concentration of acidic groups required to attain 50% relative inhibition of lysozyme by the polymers, was 10(4)--10(5) times lower than that required for the corresponding monomers. This was attributed to the multimeric nature of polymer-lysozyme binding. More than 90% lysozyme activity was recovered from egg white. Polymers exhibited reusability up to at least 16 cycles with retention of >85% recovery of specific activity from aqueous solution. In contrast, copolymer comprising natural inhibitor of lysozyme i.e. poly (N-isopropylacrylamide-co-O-acryloyl N-acetylglucosamine) lost 50% recovery of specific activity. Thermoprecipitation using these copolymers, which enables very high recovery of lysozyme from egg white, would be advantageous over pH sensitive polymers, which generally exhibit lower recovery.     

Characterization of lipase in reversed micelles formulated with cibacron blue F-3GA modified span 85

Sorbitan trioleate (Span 85) modified by Cibacron Blue F-3GA (CB) was prepared and used as an affinity surfactant to formulate a reversed micellar system for Candida rugosa lipase (CRL) solubilization. The system was characterized and evaluated by employing CRL-catalyzed hydrolysis of olive oil as a model reaction. The micellar hydrodynamic radius results reflected, to some extent, the redistribution of surfactant and water after enzyme addition, and the correlation between surfactant formulation, water content (W0), micellar size, and enzyme activity. An adequate modification density of CB was found to be important for the reversed micelles to retain enough hydration capacity and achieve high enzyme activity. Compared with the results in AOT-based reversed micelles, CRL in this micellar system exhibited a different activity behavior versus W0. The optimal pH and temperature of the encapsulated lipase remained unchanged, but the apparent activity was significantly higher than that of the native enzyme in bulk solution. Kinetic studies indicated that the encapsulated lipase in the reversed micelles of CB-formulated Span 85 followed the Michaelis-Menten equation. The Michaelis constant was found to decrease with increasing surfactant concentration, suggesting an increase of the enzyme affinity for the substrate. Stability of the lipase in the reversed micelles was negatively correlated to W0.     

Regulation and Prediction of Enzyme Activity in Reversed Micelles

The rate of cholesterol oxidation has been studied in cholesterol oxidase containing reversed micellar media consisting of the surfactant cetyltrimethylammonium bromide (CTAB), the surfactant octanol, a buffered aqueous solution, and a variety of organic solvents. By varying the composition of the medium systematically it could be deduced that the rate of cholesterol oxidation obeys the same rules as described earlier for the conversion of apolar steroids by 20β-hydroxysteroid dehydrogenase in CTAB-hexanol-organic solvent reversed micelles (Hilhorst et al. 1984). The general applicability of these rules in optimizing biocatalysis in reversed micelles is discussed.     

Spectroscopic properties of horse liver alcohol dehydrogenase in reversed micellar solutions

Catalytic and spectroscopic properties of alcohol dehydrogenase from horse liver, incorporated in reversed micellar media, have been studied. Two different reversed micellar systems have been used, one containing an anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT], the other containing a cationic (cetyltrimethylammonium bromide, CTAB) surfactant. With 1-hexanol as substrate the turnover number of the enzyme in AOT-reversed micelles is strongly dependent on the water content of the system. At low wo ([H2O]/[surfactant]) (wo less than 20) no enzymatic activity can be detected whereas at high wo (wo = 40) the turnover is only slightly lower than in aqueous solution. In CTAB-reversed micelles the dependence of the turnover number on wo is much less. The enzymatic activity is in this case significantly lower than in aqueous solution and increases only slightly with an increasing water content of the reversed micelles. Possible interactions of the protein with the surfactant interfaces in the reversed micellar media were studied via circular dichroism and fluorescence measurements. From the circular dichroism of the protein backbone it is observed that the protein secondary structure is not significantly affected upon incorporation in the reversed micelles since the far-ultraviolet spectrum is not altered. Results from time-resolved fluorescence anisotropy experiments indicate that, especially in AOT-reversed micelles, interactions between the protein and the surfactant interface are largely electrostatic in nature, as evident from the dependence on the pH of the buffer used. In CTAB-reversed micellar solutions such interactions appear to be much less pronounced than in AOT.     

One-step purification of proteins from chicken egg white using counter-current chromatography

Proteins present in chicken egg white are separated by counter-current chromatography (CCC) in one step using a cross-axis coil planet centrifuge (X-axis CPC). The separation was performed with an aqueous polymer two-phase system composed of 16% (w/w) poly(ethylene glycol) 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 1.0 ml/min. From about 20 g of the crude egg white solution, lysozyme, ovalbumin, and ovotransferrin were resolved within 5.5 h. Each component was identified by 12% SDS gel electrophoresis with Coomassie brilliant blue staining.     

Regulation and Prediction of Enzyme Activity in Reversed Micelles

The rate of cholesterol oxidation has been studied in cholesterol oxidase containing reversed micellar media consisting of the surfactant cetyltrimethylammonium bromide (CTAB), the surfactant octanol, a buffered aqueous solution, and a variety of organic solvents. By varying the composition of the medium systematically it could be deduced that the rate of cholesterol oxidation obeys the same rules as described earlier for the conversion of apolar steroids by 20β-hydroxysteroid dehydrogenase in CTAB-hexanol-organic solvent reversed micelles (Hilhorst et al. 1984). The general applicability of these rules in optimizing biocatalysis in reversed micelles is discussed.     

Novel function of guanidine hydrochloride in reverse micellar extraction of lysozyme from chicken egg white

The efficiency of guanidine hydrochloride (GuHCl) addition in the suppression of gel formation and the extraction of lysozyme during reverse micellar extraction from chicken egg white was investigated. A low concentration of GuHCl in the feed permitted the successful extraction of lysozyme in its native form without gel formation, which is perceived as a novel function of GuHCl. The highest recovery and specific activity of lysozyme were obtained at a GuHCl concentration of 0.06 M in 25 mM AOT reverse micellar extraction from 20-fold-diluted natural chicken egg white. Lysozyme and ovalbumin CD spectra in the corresponding GuHCl aqueous solutions revealed no changes in the higher order structures of the proteins. Furthermore, the specific activity of lysozyme in the feed was well preserved in the GuHCl system. (c) 1995 John Wiley & Sons, Inc.     

Protein refolding by reversed micelles utilizing solid-liquid extraction technique

This article reports that a reversed micellar solution is useful for refolding proteins directly from a solid source. The solubilization of denatured RNase A, which had been prepared by reprecipitation from the denaturant protein solution, into reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) has been investigated by a solid-liquid extraction system. This method is an alternative to the ordinary protein extraction in reversed micelles based on the liquid-liquid extraction. The solid-liquid extraction method was found to facilitate the solubilization of denatured proteins more efficiently in the reversed micellar media than the ordinary phase transfer method of liquid extraction. The refolding of denatured RNase A entrapped in reversed micelles was attained by adding a redox reagent (reduced and oxidized glutathion). Enzymatic activity of RNase A was gradually recovered with time in the reversed micelles. The denatured RNase A was completely refolded within 30 h. In addition, the efficiency of protein refolding was enhanced when reversed micelles were applied to denatured RNase A containing a higher protein concentration that, in the case of aqueous media, would lead to protein aggregation. The solid-liquid extraction technique using reversed micelles affords better scale-up advantages in the direct refolding process of insoluble protein aggregates.     

The effect of demulsifiers on lysozyme extraction from hen egg white using reverse micelles

The liquid–liquid extraction of protein from buffered aqueous phases using reverse micelles (RM) has been extensively researched from a fundamental point of view. However, very little effort has been expended at scaling up this process for the extraction of real fermentation broth. When real broths are used with reverse micellar phases there are major problems with emulsion formation. In this study the effect of a variety of demulsifiers on lysozyme extraction was evaluated in terms of their influence on the separating properties of the emulsion, water content (W _o ), and, extraction yield and kinetics from both buffer and hen egg white. In addition, the use of a low shear contactor (a Graesser or `raining bucket') was assessed in terms of its suitability as a RM contactor. It was found that most of the demulsifiers reduced the settling time of the emulsion, and enhanced the yield and kinetics of lysozyme extraction from hen egg white. It was hypothesised that this was due to the demulsifier displacing the lysozyme from the interface and preventing the protein unfolding and precipitating. This effect was found to depend on both the generic type of demulsifier, and its concentration.     

Separation of lysozyme using superparamagnetic carboxymethyl chitosan nanoparticles

Functionalized Fe(3)O(4) nanoparticles conjugated with polyethylene glycol (PEG) and carboxymethyl chitosan (CM-CTS) were developed and used as a novel magnetic absorbing carrier for the separation and purification of lysozyme from the aqueous solution and chicken egg white, respectively. The morphology of magnetic CM-CTS nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of superparamagnetic carboxymethyl chitosan nanoparticles (Fe(3)O(4) (PEG+CM-CTS)) was about 15 nm, and could easily aggregate by a magnet when suspending in the aqueous solution. The adsorption capacity of lysozyme onto the superparamagnetic Fe(3)O(4) (PEG+CM-CTS) nanoparticles was determined by changing the medium pH, temperature, ionic strength and the concentration of lysozyme. The maximum adsorption loading reached 256.4 mg/g. Due to the small diameter, the adsorption equilibrium of lysozyme onto the nanoparticles reached very quickly within 20 min. The adsorption equilibrium of lysozyme onto the superparamagnetic nanoparticles fitted well with the Langmuir model. The nanoparticles were stable when subjected to six repeated adsorption-elution cycles. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The lysozyme was purified from chicken egg white in a single step had higher purity, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Considering that the superparamagnetic nanoparticles possess the advantages of high efficiency, cost-effectiveness and excellent binding of a larger amount of lysozyme and easier separation from the reaction system, thus this type of superparamagnetic nanoparticles would bring advantages to the conventional separation techniques of lysozyme from chicken egg white.     

Studies on the extraction and back-extraction of a recombinant cutinase in a reversed micellar extraction process

This work deals with the extraction and back-extraction of a recombinant cutinase using AOT reversed micelles in isooctane. The effect of pH, ionic strength, AOT concentration and temperature on the extraction and back-extraction of the cutinase was investigated. High extraction (97%) of the cutinase was achieved at pH 7.0 with a 50 mM Tris-HCl buffer solution containing 100 mM KCl, but a low activity was detected in the reversed micellar phase. At pH 9.0, cutinase was extracted (75%) to the reversed micelles with higher activity. Cutinase was recovered (50%) from a reversed micellar phase (100 mM AOT/isooctane) into a 50 mM Tris-HCl buffered solution at pH 9.0 with 100 mM KCl, and 20°C. Protein and cutinase activity global yields of 38 and 45%, respectively, were obtained for the global process, extraction and back-extraction steps, using low ionic strength, pH 9.0, 100 mM AOT and 20°C.Maria das Graças Carneiro da Cunha acknowledges a Ph.D. fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Centro de Pesquisas Aggeu Magalhães, Brasil. This work was partly financed by the BRIDGE Programme (Contract BIOT-CT91-0274(DTEE)).     

Protein refolding in reversed micelles

A novel process has been developed which uses reversed micelles to isolate denatured protein molecules from each other and allows them to refold individually. These reversed micelles are aqueous phase droplets stabilized by the surfactant AOT and suspended in isooctane. By adjusting conditions such that only one protein molecule is present per reversed micelle, it was possible to achieve independent folding without encountering the problem of aggregation due to interactions with neighboring molecules. The feasibility of this process was demonstrated using bovine pancreatic ribonuclease A as a model system. It was shown that denatured and reduced ribonuclease can be transferred from a buffered solution containing guanidine hydrochloride into reversed micelles to a greater extent than native enzyme under the same conditions. The denaturant concentration can then be significantly reduced in the reversed micellar phase, while retaining most of the protein, by means of extractive contacting stages with a denaturant-free aqueous solution. Denatured and reduced ribonuclease will subsequently recover full activity inside reversed micelles within 24 h upon addition of a mixture of reduced and oxidized glutathione to reoxidize disulfide bonds. Extraction of this refolded enzyme from reversed micelles back into aqueous solution can be accomplished by contacting the reversed micelle phase with a high ionic strength (1.0M KCl) aqueous solution containing ethyl acetate.     

Polarographic measurement of oxygen uptake using lipoxygenase in reverse micelles

Lipoxygenase-catalyzed linoleic acid peroxidation was chosen as a model system to study the applicability of oxygraphy to monitor the oxygen uptake in organic solvents containing reverse micelles. Care was taken to control the oxygen back transfer from the atmosphere to the sample micellar solution, resulting in a significant improvement of electrode response. Under these conditions, lipoxygenase activity was linear up to 100 mug of enzyme. Given the quality of the calibration curve and the good correlation between lipoxygenase and ascorbate oxidase, the described technique is proposed as an alternative method for determining lipoxygenase activity in reverse micelles. The reliability of this technique was confirmed by the good agreement between polarography and classic spectrophotometry in kinetic studies. Preliminary experiments carried out on soybean cells solubilized in a Tween 85-isopropylpalmitate system demonstrated that a light-dependent oxygen uptake can be measured. The authors propose that the Clark-type electrode be employed to study both the activity of oxidasic enzymes in reverse micelles and cell viability and physiology in organic solvents.     

Formation of biocompatible reversed micellar systems using phospholipids

Formation of reversed micellar systems using biocompatible components was revealed by a significant increase of water content in the organic phase. Soybean lecithin (SL), which is a mixture of different phospholipids, and phosphatidylcholine (PC) purified from soybean were used as the amphiphilic molecule. Fatty acid and fatty acid ethyl esters were used as the organic solvent. Reversed micelles were formed in the following combinations of (amphiphilic molecule)/(organic solvent): SL/ethyl caproate, SL/ethyl oleate, SL/ethyl linoleate, PC/ethyl caproate, and PC/oleic acid. Characterization of the micelles using small angle X-ray scattering analysis was presented. Reversed micelles formed in SL/ethyl caproate, SL/ethyl oleate, and PC/ethyl caproate systems were spherical. Their radius of gyration was about 40? when the water concentration in the organic phase was maximal. Maximal water concentrations in SL/ethyl caproate and PC/ethyl caproate reversed micellar systems decreased with increasing salt concentration in the aqueous phase. Micelle sizes also decreased with increased salt concentration. The extraction of protein cytochrome c using the reversed micellar system was demonstrated. Application of these reversed micellar systems will expand to pharmaceutical and food industries.