Study of the factors affecting the extraction of soybean protein by reverse micelles

In this work, the forward and back extraction of soybean protein by reverse micelles was studied. The reverse micellar systems were formed by anionic surfactant sodium bis(2-ethyl hexyl) sulfosuccinate (AOT), isooctane and KCl solution. The effects of AOT concentration, aqueous pH, KCl concentration and phase volume ratio on the extraction efficiency of soybean protein were tested. Suitability of reverse micelles of AOT and Triton-X-100/AOT mixture in organic solvent toluene for soybean protein extraction was also investigated. The experimental results lead to complete forward extraction at the AOT concentration 120 mmol l⁻¹, aqueous pH 5.5 and KCl concentration 0.8 mol l⁻¹. The backward extraction with aqueous phase (pH 5.5) resulted in 100% extraction of soybean protein from the organic phase.     

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)).     

Extraction and purification of a lectin from small black kidney bean (Phaseolus vulgaris) using a reversed micellar system

Lectin from crude extract of small black kidney bean (Phaseolus vulgaris) was successfully extracted using the reversed micellar extraction (RME). The effects of water content of organic phase (W_o), ionic strength, pH, Aerosol-OT (AOT) concentration and extraction time on the forward extraction and the pH and ionic strength in the backward extraction were studied to optimize the extraction efficiency and purification factor. Forward extraction of lectin was found to be maximum after 15 min of contact using 50 mM AOT in organic phase with W_o 27 and 10 mM citrate-phosphate buffer at pH 5.5 containing 100 mM NaCl in the aqueous phase. Lectin was backward extracted into a fresh aqueous phase using sodium-phosphate buffer (10 mM, pH 7.0) containing 500 mM KCl. The overall yield of the process was 53.28% for protein recovery and 8.2-fold for purification factor. The efficiency of the process was confirmed by gel electrophoresis analysis.     

Process optimization for reverse micellar extraction of stem bromelain with a focus on back extraction

In the current study, reverse micellar extraction (RME) for the purification of stem bromelain was successfully achieved using the sodium bis(2‐ethylhexyl) sulfosuccinate (AOT)/isooctane system. A maximum forward extraction efficiency of 58.0% was obtained at 100 mM AOT concentration, aqueous phase pH of 8.0 and 0.2 M NaCl. Back extraction studies on altering stripping phase pH and KCl concentration, addition of counter‐ion and iso‐propyl alcohol (IPA) and mechanical agitation with glass beads indicated that IPA addition and agitation with glass beads have significant effects on extraction efficiency. The protein extraction was higher (51.9%) in case of the IPA (10% v/v) added system during back extraction as compared to a cetyltrimethylammonium bromide (100 mM) added system (9.42%). The central composite design technique was used to optimize the back extraction conditions further. Concentration of IPA, amount of glass beads, mixing time, and agitation speed (in rpm) were the variables selected. IPA concentration of 8.5% (v/v), glass bead concentration of 0.6 (w/v), and mixing time of 45 min at 400 rpm resulted in higher back extraction efficiency of 45.6% and activity recovery of 88.8% with purification of 3.04‐fold. The study indicated that mechanical agitation using glass beads could be used for destabilizing the reverse micelles and release of bromelain back into the fresh aqueous phase. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:845–855, 2014     

AOT/异辛烷反胶束萃取纯化胰蛋白酶及酶变性失活问题的解决

用反胶束技术分离纯化蛋白质,具有高选择性、易于大规模操作等优点,具有良好的工业应用前景。但是离子型表面活性剂形成的反胶束体系萃取蛋白质容易引起蛋白质的变性,这是由于离子型表面活性剂的强电荷作用所导致的。对用AOT/异辛烷反胶束体系从胰酶粗提物中萃取胰蛋白酶进行了研究,通过在反胶束相加入乙醇,解决了反胶束萃取蛋白质时蛋白质变性失活的问题。并且由于乙醇的加入大大减少了分相的时间,简化了实验步骤,优化了实验方法,使此技术在工业上的大规模应用成为可能。通过优化各种实验条件,胰蛋白酶的前萃取率达到90%,反萃取率接近100%。最终得率为88%。纯化后的比活提高了5倍多,从300U/mg左右提高到了1800U/mg。     

Forward and backward extraction rates of amino acid in reversed micellar extraction

The mass transfer characterization in reversed micellar extraction of amino acid phenylalanine (Phe) is presented. The mass transfer rates in forward extraction of Phe from aqueous KCl solutions (pH 1.4 – 2.3) to AOT/isooctane reversed micellar solutions and in backward extraction from the reversed micellar organic phase to KHCO₃/KOH buffer solutions (pH 9.0 – 11.0) were investigated using a stirred cell with a flat liquid–liquid interface. Both the forward and the backward extraction rates are controlled by the interfacial rate processes, i.e., the solubilization and the release processes. The solubilizing rate constants for the forward extraction of Phe increase with decreasing pH and initial Phe concentration and with increasing initial AOT concentration. On the other hand, the releasing rate constants for the backward extraction decrease with increasing initial AOT concentration and with decreasing ionic strength, but are little influenced by pH. The backward extraction rates are fairly slow compared to the forward extraction rates, and are accelerated by the addition of 2-methyl-2-propanol, similar to the extraction of protein lysozyme.     

Halophilic amylase from a moderately halophilic Micrococcus

A moderately halophilic Micrococcus sp., isolated from unrefined solar salt, produced a considerable amount of extracellular dextrinogenic amylase when cultivated aerobically in media containing 1 to 3 m NaCl. The Micrococcus amylase had maximal activity at pH 6 to 7 in 1.4 to 2 m NaCl or KCl at 50 C. Calcium ion and a high concentration of NaCl or KCl were essential for activity and stability of the amylase. The salt response of the amylase depended greatly on the pH and temperature of the enzyme assay.     

Important parameters affecting efficiency of protein refolding by reversed micelles

Refolding of denatured RNase A as a model of inclusion bodies was performed by reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) in isooctane. In the novel refolding process, a solid-liquid extraction was utilized as an alternative to the ordinary protein extraction by reversed micelles based on a liquid-liquid extraction. First, the effects of operational parameters such as concentration of AOT, W(o) (= [H(2)O]/[AOT]), and pH were examined on the solubilization of solid denatured proteins into a reversed micellar solution. The solubilization was facilitated by a high AOT concentration, a high W(o) value, and a high pH in water pools. These conditions are favorable for the dispersion of the solid protein aggregates in an organic solvent. Second, the renaturation of the denatured RNase A solubilized into the reversed micellar solution was conducted by addition of glutathione as a redox reagent. A complete renaturation of RNase A was accomplished by adjusting the composition of the redox reagent even at a high protein concentration in which protein aggregation would usually occur in aqueous media. In addition, the renaturation rates were improved by optimizing water content (W(o)) and the pH of water pools in reversed micelles. Finally, the recovery of renatured RNase A from the reversed micellar solution was performed by adding a polar organic solvent such as acetone into the reversed micellar solution. This precipitation method was effective for recovering proteins from reversed micellar media without any significant reduction in enzymatic activity.     

Extraction of monoclonal antibodies (IgG1) using anionic and anionic/nonionic reverse micelles

Purification schemes for antibody production based on affinity chromatography are trying to keep pace with increases in cell culture expression levels and many current research initiatives are focused on finding alternatives to chromatography for the purification of Monoclonal antibodies (MAbs). In this article, we have investigated an alternative separation technique based on liquid–liquid extraction called the reverse micellar extraction. We extracted MAb (IgG1) using reverse micelles of an anionic surfactant, sodium bis 2‐ethyl‐hexyl sulfosuccinate (AOT) and a combination of anionic (AOT) and nonionic surfactants (Brij‐30, Tween‐85, Span‐85) using isooctane as the solvent system. The extraction efficiency of IgG1 was studied by varying parameters, such as pH of the aqueous phase, cation concentration, and type and surfactant concentration. Using the AOT/Isooctane reverse micellar system, we could achieve good overall extraction of IgG1 (between 80 and 90%), but only 30% of the bioactivity of IgG1 could be recovered at the end of the extraction by using its binding to affinity chromatography columns as a surrogate measure of activity. As anionic surfactants were suspected as being one of the reasons for the reduced activity, we decided to combine a nonionic surfactant with an anionic surfactant and then study its effect on the extraction efficiency and bioactivity. The best results were obtained using an AOT/Brij‐30/Isooctane reverse micellar system, which gave an overall extraction above 90 and 59% overall activity recovery. An AOT/Tween‐85/Isooctane reverse micellar system gave an overall extraction of between 75 and 80% and overall activity recovery of around 40–45%. The results showed that the activity recovery of IgG1 can be significantly enhanced using different surfactant combination systems, and if the recovery of IgG1 can be further enhanced, the technique shows considerable promise for the downstream purification of MAbs. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Development of a new reversed micelle liquid emulsion membrane for protein extraction

A new type of liquid emulsion membrane containing reversed micelles for protein extraction is introduced. A three-step extraction mechanism is proposed including solubilization, transportation, and release of the protein. The surfactants Span80 and sodium di(2-ethylhexyl)sulfosuccinate (AOT) are used to stabilize the membrane phase and to build up the reversed micelles, respectively. alpha-Chymotrypsin was used as the model protein. The condition in the internal phase inhibits the solubilization process of the already extracted protein back into reversed micelles. Concerning the solubilization, we studied the influence of the AOT concentration in the membrane phase and the ionic strength in the external phase. The extraction rate increases with higher AOT concentration and decreases with higher ionic strength. Using NaCl in the external phase led to better extraction results than using KCl. Maximum extraction results of 98% into the membrane phase and 65% into the internal phase were obtained. This condition retained 60% of the enzyme's activity. The concentration of KCl in the internal phase does not affect the solubilization rate but the release into the internal phase. By this way the ionic strength in the internal phase is used as the driving force for the protein release. The solubilization process is much faster than the diffusion and the releasing process, as found by variation of the extraction time. The influence of the operating conditions on the membrane swelling is also discussed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 267-273, 1997.     

Selective separation and purification of two lipases from chromobacterium viscosum using AOT reversed micelles

Selective separation and purification of two lipases form Chromobacterium viscosum were carried out by liquid-liquid extraction using a reversed micellar system. Optimum parameters for extraction were determined using a 250 mM AOT micellar solution in isooctane. Complete separation of the two lipases was achieved at pH 6.0 with a 50mM potassium phosphate buffer solution containing 50 mM KCI. By adding 2.5% by volume of ethanol to the lipase-loaded micellar solution, 85% of the extracted lipase could be recovered in a new aqueous phase, 50 mM K(2)HPO(4) with 50 mM KCl, at pH 9.0. Lipase A was purified 2.6-fold with a recovery of 86%, and lipase B by 1.5-fold with a recovery of 76%.     

Downstream processing of soy hull peroxidase employing reverse micellar extraction

Phase transfer studies were conducted to evaluate the solubilization of soy hull peroxidase (SHP) in reverse micelles formed in isooctane/butanol/hexanol using the cationic surfactant cetyltrimethylammonium bromide (CTAB). The effect of various parameters such as pH, ionic strength, surfactant concentration of the initial aqueous phase for forward extraction and buffer pH, type and concentration of salt, concentration of isopropyl alcohol and volume ratio for back extraction was studied to improve the efficiency of reverse micellar extraction. The active SHP was recovered after a complete cycle of forward and back extraction. A forward extraction efficiency of 100%, back extraction efficiency of 36%, overall activity recovery of 90% and purification fold of 4.72 were obtained under optimised conditions. Anionic surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) did not yield good results under the conditions studied. The phase transfer of soy hull peroxidase was found to be controlled by electrostatic and hydrophobic interactions during forward and back extraction respectively.     

Electrostatic immobilization of pectinase on negatively charged AOT-Fe3O4 nanoparticles

Enzyme immobilization on magnetic nanoparticles (MNPs) has been a field of intense studies in biotechnology during the past decade. The present study suggests MNPs negatively charged by docusate sodium salt (AOT) as a support for pectinase immobilization. AOT is a biocompatible anionic surfactant which can stabilize MNPs. Electrostatic adsorption can occur between enzyme with positive charge and oppositely charged surface of MNPs (ca. 100 nm). The effect of three factors, i.e. initial enzyme concentration, aqueous pH and AOT concentration in different levels was investigated on pectinase immobilization. Maximum specific activity (1.98 U/mg enzyme) of immobilized pectinase and maximum enzyme loading of 610.5 mg enzyme/g support was attained through the experiments. Initial enzyme concentration is significantly important on both loading and activity of immobilized enzyme, while pH and AOT concentration only affect the amount of immobilized enzyme. Immobilized enzyme on MNPs was recovered easily through magnetic separation. At near pH of immobilization, protein leakage in reusability of immobilized enzyme was low and activity loss was only 10–20% after six cycles. Since pH is associated with immobilization by electrostatic adsorption, the medium pH was changed to improve the release of protein from the support, as well. MNPs properties were investigated using Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FT-IR) spectroscopy, and Dynamic Light Scattering (DLS) analysis.     

Separation and purification of periplasmic cytochrome c553 using reversed micelles

Surfactant concentration, ionic strength, and pH were optimised for the selective separation and purification of periplasmic cytochrome c553 from recombinant E. coli TG2 cells using response surface methodology. Back-extraction was accomplished using counter-ionic surfactant addition. Optimum forward extraction conditions were: 65 mM bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT), 0.07 M NaCl, and pH 8.4, while the optimum back-extraction conditions were 80 mM trioctylmethylammonium chloride, 0.85 M KCl, and pH 9.62. In comparison to a conventionally purified sample using column chromatography (10 mg cytochrome c553 l–1 with a purity of 0.66), reverse micelles achieved the same concentration and similar purity (0.50) in only two simple steps.     

Solubilizing water involved in protein extraction using reversed micelles

The extraction of protein using reversed micelles was investigated in relation to the amount of solubilizing water in the reversed micellar organic phase. The minimal concentration of amphiphilic molecule di-2-ethylhexyl sodium sulfosuccinate (C(20)H(37)O(7)Na) (AOT) required for 100% cytochrome c extraction was recognized. This critical AOT concentration increased with protein concentration in the aqueous phase. On this minimal AOT condition, the molar ratio of solubilizing water to extracted protein was found to be a constant of 3500 under C(KCI) = 1.0 x 10(2) mol . m(-3) in this system. This ratio means the hydrophillic surroundings required for extracting one protein molecule into the micellar organic phase under the suitable pH and salt concentration for the forward extraction. In this regard, AOT molecules seemed to take the part of water solubilizing agent in the reversed micellar extraction. This role of AOT is important to extract protein under the suitable pH and salt concentration. The amount of solubilizing water in the protein-containing system was larger than in the protein-free system. This difference shows that the water molecules accompany the extracted protein into the reversed micellar organic phase at constant ratio 2200 under C(KCI) = 1.0 x 10(2) mol . m(-3), i.e., accompanying water molecules per one extracted protein. The minimal AOT concentration increased with ionic strength. On this minimal AOT condition, the molar ratio of solubilizing water to extracted protein also increased with ionic strength, so that in higher ionic strength, more solubilizing water was required. Then more AOT was required to provide the hydrophillic surroundings for protein. The pH affected the minimal AOT concentration required for 100% protein extraction.     

Production of amylase by newly isolated moderate halophile,Halobacillus sp. strain MA-2

Production of extracellular amylase was demonstrated under stress conditions of high temperature and high salinity in aerobically cultivated culture of a newly isolated moderately halophilic bacterium of spore-forming Halobacillus sp. strain MA-2 in medium containing starch, peptone, beef extract, and NaCl. The maximum amylase production was secreted in the presence of 15% (w/v) Na(2)SO(4) (3.2 U ml(-1)). The isolate was capable of producing amylase in the presence of NaCl, NaCH(3)COOH, or KCl, with the results NaCl>NaCH(3)COOH>KCl. Maximum amylase activity was exhibited in the medium containing 5% (w/v) NaCl (2.4 U ml(-1)). Various carbon sources induced enzyme production. The potential of different carbohydrates in the amylase production was in the order: dextrin>starch>maltose>lactose>glucose>sucrose. In the presence of sodium arsenate (100 mM), maximum production of the enzyme was observed at 3.0 U ml(-1). Copper sulfate (0.1 mM) decreased the amylase production considerately, while lead nitrate had no significant enhancement on amylase production (p<0.05). The pH, temperature, and aeration optima for enzyme production were 7.8, 30 degrees C, and 200 rpm, respectively, while the optimum pH and temperature for enzyme activity was 7.5-8.5 and 50 degrees C, respectively.     

Reverse micellar extraction of papain with cationic detergent based system: An optimization approach

In this study, reverse micellar extraction of papain model system was performed using cetyltrimethylammonium bromide (CTAB)/iso-octane/hexanol/butanol system to optimize the forward and back extraction efficiency (BEE). A maximum forward extraction efficiency of 55.0, 61.0, and 54% was achieved with an aqueous phase pH of 11.0, 150?mM CTAB/iso-octane and 0.1?M NaCl, respectively. Taguchi’s orthogonal array was applied to optimize the pH of stripping phase, concentration of isopropyl alcohol (IPA) and potassium chloride (KCl) for maximizing BEE. The optimal levels of stripping phase pH, concentration of IPA and KCl were found to be 6, 20% (v/v), and 0.8?M, respectively. Under these optimal levels, the BEE was found to be 88% after which enzyme activity was recovered with 2.5-fold purification. Further optimization was performed using artificial neural network-linked genetic algorithm, where the BEE was improved to 90.52% with pH 6, IPA (%)?=?19.938, and KCl (M)?=?0.729.     

Reverse micelles in protein separation: the use of silica for the back-transfer process

In order to use reverse micellar solutions successfully for the separation of proteins, good methods are needed to recover the biomolecules into an aqueous environment after solubilization into organic micellar media. Usually the recovery is accomplished by equilibrating the protein-loaded reverse micellar solution with a water phase containing an appropriate salt (back-transfer). In this article we describe an alternative "back extraction" procedure which is based on the addition of silica to the protein-containing reverse micellar solution. In this way, the water is stripped from the reverse micellar solution. [i.e., bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane/water] and the proteins adsorb to the silica particles. The adsorption process is shown to be practically quantitative. The subsequent recovery of the proteins form the silica into an aqueous solution turns out to be most efficient at alkaline pH (pH 8); 60-80 of the total protein (alpha-chymotrypsin or trypsin) could be recovered. The specific enzyme activity at the end of the whole cycle can be as high as 80-100%. The procedure is applied also for the back extraction from micellar solutions in which, instead of AOT, a biocompatible surfactant such as a synthetic short-chain lecithin was used. It is shown that the recovery of a alpha-chymotrypsin and trypsin is also achievable under these conditions in quite good yield and under good maintenance of the enzyme's catalytic activity. (c) 1993 John Wiley & Sons, Inc.     

Aqueous two phase extraction of invertase from baker's yeast: Effect of process parameters on partitioning

Invertase (β-d-fructofuronoside fructohydrolase) is an industrially important enzyme useful for the hydrolysis of sucrose. The potential of aqueous two phase extraction for the isolation and purification of invertase from crude baker's yeast is explored. Influence of the process parameters such as type of phase forming salts, PEG molecular weight, concentration of salt and polymer, tie line length and volume ratio on partitioning of invertase was studied. PEG 3350/magnesium sulphate system was found most suitable for the extraction which has resulted in favorable pH (5 ± 0.2) for the enzyme extraction. Polymer and salt concentration were found to significantly affect the degree of purification and enzyme recovery of invertase. The purity of ∼8.81 fold was obtained compared to crude extract with recovery of 77% at the standardized process conditions. Overall results demonstrated the feasibility of aqueous two phase extraction for the isolation and purification of invertase without the need of multiple steps.     

Study of the Stability Of Vaccinium myrtillus Peroxidase in Reverse Micellar Systems

The stability of a cationic peroxidase isolated and purified from a cell suspension of Vaccinium myrtillus, microencapsulated in reverse micelles of sodium dioctylsulfosuccinate (AOT) was evaluated. By using a central composite design (CCD), some relevant parameters for the enzymatic activity, such as surfactant and water concentration, pH and buffer molarity, were analysed. The response surface curves showed that 50 mM AOT, 500 mM water, 80 mM buffer and pH 7.6 were the best conditions for enzyme stability. The effect of carbohydrates and polyols on enzyme stability was also evaluated. At 20 mM, carbohydrates like arabinose, and trehalose increased the enzymatic stability by a factor of 4.4 and 2.3, respectively, but melezitose had no effect. From the three polyols tested, inositol and sorbitol increased the peroxidase stability by a factor of 3.8 and 1.8, respectively, while mannitol had no effect.