Solubilization of enzymes in water-in-oil microemulsions and their rapid and efficient release through use of a pH-degradable surfactant

α-Chymotrypsin and lysozyme were solubilized in a water/O-[(2-tridecyl, 2-ethyl-1,3-dioxolan-4-yl)methoxy]–O′-methoxy poly(ethylene glycol) (CK-2,13 surfactant)/isooctane water-in-oil microemulsion solution at 1.5–2 and 10 g l⁻¹ for 0.15 and 1.2 M CK-2,13, respectively. Upon contact with an equal volume of 0.1 M NaH₂PO₄/Na₂HPO₄ buffer, pH 5, a three-phase system (Winsor-III system) was formed, consisting of a surfactant-rich middle phase and aqueous and isooctane-rich “excess” phases. Both enzymes were rapidly released into the aqueous excess phase, with 70% recovery of each in 30 and 60 min for microemulsion solutions containing 0.15 and 1.2 M surfactant, respectively. The recovered enzymes retained >90% of their original specific activity.     

Extraction of bovine serum albumin using nanoparticulate reverse micelles

The extraction of a relatively large molecular weight protein, bovine serum albumin (BSA), using nano-sized reverse micelles of nonionic surfactant polyoxyethylene p-t-octylphenol (Triton-X-100) is attempted for the first time. Suitability of reverse micelles of anionic surfactant sodium bis (2-ethyl hexyl) sulfosuccinate (AOT) and Triton-X-100/AOT mixture in organic solvent toluene for BSA extraction is also investigated. Although, the size of the Triton-X-100 reverse micelle in toluene is large enough to host BSA molecule in the hydraulic core, the overall extraction efficiency is found to be low, which may be due to lack of strong driving force. AOT/toluene system resulted in complete forward extraction at aqueous pH 5.5 and a surfactant concentration of 160 mM. The back extraction with aqueous phase (pH 5.5) resulted in 100% extraction of BSA from the organic phase. The addition of Triton-X-100 to AOT reduced the extraction efficiency of AOT reverse micelles, which may be attributed to reduced hydrophobic interaction. The circular dichroism (CD) spectrum of BSA extracted using AOT/toluene reverse micelles indicated the structural stability of the protein extracted.     

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.     

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.     

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     

A Novel α‐MoO3/Single‐Walled Carbon Nanohorns Composite as High‐Performance Anode Material for Fast‐Charging Lithium‐Ion Battery

Orthorhombic α‐MoO₃ is a potential anode material for lithium‐ion batteries due to its high theoretical capacity of 1100 mAh g^?1 and excellent structural stability. However, its intrinsic poor electronic conductivity and high volume expansion during the charge–discharge process impede it from achieving a high practical capacity. A novel composite of α‐MoO₃ nanobelts and single‐walled carbon nanohorns (SWCNHs) is synthesized by a facile microwave hydrothermal technique and demonstrated as a high‐performance anode material for lithium‐ion batteries. The α‐MoO₃/SWCNH composite displays superior electrochemical properties (654 mAh g^?1 at 1 C), excellent rate capability (275 mAh g^?1 at 5 C), and outstanding cycle life (capacity retention of >99% after 3000 cycles at 1 C) without any cracking of the electrode. The presence of SWCNHs in the composite enhances the electrochemical properties of α‐MoO₃ by acting as a lithium storage material, electronic conductive medium, and buffer against pulverization.     

A study on the interaction between cadmium and α‐chymotrypsin and the underlying mechanisms

Because cadmium might interact with proteins and, thus, exert toxicity in organisms, it is vital to understand the molecular mechanism of the interaction between cadmium and biologically relevant proteins as well as the structural and functional changes in these proteins. In this study, the interaction between α‐chymotrypsin (α‐ChT) and cadmium chloride (CdCl₂) was investigated by performing enzyme activity determinations, multispectroscopic measurements, isothermal titration calorimetry, and molecular docking studies. It was demonstrated that CdCl ₂ binds to α‐ChT mainly via electrostatic forces with (21.0 ± 0.982) binding sites, leading to the increase of α‐helix and the decrease of β‐sheet. The interaction between CdCl ₂ and α‐ChT loosened the protein skeleton and increased the molecular volume of α‐ChT. CdCl ₂ first binds to the interface of α‐ChT and then interacts with the key residues His 57 or Asp 102 or both in the active sites, leading to the activity inhibition of α‐ChT under the exposure of high CdCl ₂ concentrations.     

Mechanism of extraction of chymotrypsin into isooctane at very low concentrations of aerosol OT in the absence of reversed micelles

Chymotrypsin is easily extracted from an aqueous solution into isooctane containing the anionic surfactant aerosol OT (AOT). The concentration of AOT needed to efficiently extract 0.5 mg/mL CMT is as low as 1 mM and as low as 0.2 mM AOT was sufficient to extract the protein into isooctane. The extraction process was unaffected by 10% (v/v) ethyl acetate in the isooctane phase. Moreover, spectroscopic analysis by electron paramagnetic resonance indicated that CMT did not exist inside a discreet water pool of a reversed micelle. Calculations of the number of AOT molecules associated per extracted CMT molecule indicate that only ca. 30 surfactant molecules interact with the protein, a value too low for reversed micellar incorporation of the protein in isooctane. These studies suggested that reversed micelles do not need to be involved in the actual transfer of the protein from the aqueous to the organic phase and protein solubilization in the organic phase is possible in the absence of reversed micelles. Based on these findings, a new mechanism has been proposed herein for protein extraction via the phase transfer method involving ionic surfactants. The central theme of this mechanism is the formation of an electrostatic complex between CMT and AOT at the aqueous/organic interface between AOT and CMT, thereby leading to the formation of a hydrophobic species that partitions into the organic phase. Consistent with this mechanism, the efficiency of extraction is dependent on the interfacial mass transfer, the concentrations of CMT and AOT in the aqueous and organic phases, respectively; the ionic strength of the aqueous phase; and the presence of various cosolvents. (c) 1994 John Wiley & Sons, Inc.     

Backward extraction of reverse micellar encapsulated proteins using a counterionic surfactant

The back-extraction of proteins encapsulated in AOT reverse micelles was performed by adding a counterionic surfactant, either TOMAC or DTAB. This novel backward transfer method gave higher backward extraction yields compared to the conventional method with high salt and high pH of the aqueous stripping solution. The protein activity was maintained in the resulting aqueous phase, which in this case had a near neutral pH and low salt concentration. A sharp decrease of the water content was observed in the organic phase corresponding to protein back-extraction using TOMAC. The backward transfer mechanism was postulated to be caused by electrostatic interaction between oppositely charged surfactant molecules, which lead to the collapse of the reverse micelles. The back-extraction process with TOMAC was found to be very fast; more than 100 times faster than back-extraction with the conventional method, and as much as 3 times faster than forward extraction. The formation of 1:1 complexes of AOT and TOMAC in the solvent phase was observed, and these hydrophobic complexes could be efficiently removed from the solvent using adsorption onto Montmorillonite in order for the organic solvent to be reused. A second cationic surfactant, DTAB, confirmed the general applicability of counterionic surfactants for the backward transfer of proteins.     

Spectroscopic and molecular modelling analysis of the interaction between ethane‐1,2‐diyl bis(N,N‐dimethyl‐N‐hexadecylammoniumacetoxy)dichloride and bovine serum albumin

Several spectroscopic approaches namely fluorescence, time‐resolved fluorescence, UV‐visible, and Fourier transform infra‐red (FT‐IR) spectroscopy were employed to examine the interaction between ethane‐1,2‐diyl bis(N,N‐dimethyl‐N‐hexadecylammoniumacetoxy)dichloride (16‐E2‐16) and bovine serum albumin (BSA). Fluorescence studies revealed that 16‐E2‐16 quenched the BSA fluorescence through a static quenching mechanism, which was further confirmed by UV–visible and time‐resolved fluorescence spectroscopy. In addition, the binding constant and the number of binding sites were also calculated. The thermodynamic parameters at different temperatures (298 K, 303 K, 308 K and 313 K) indicated that 16‐E2‐16 binding to BSA is entropy driven and that the major driving forces are electrostatic interactions. Decrease of the α‐helix from 53.90 to 46.20% with an increase in random structure from 22.56 to 30.61% were also observed by FT‐IR. Furthermore, the molecular docking results revealed that 16‐E2‐16 binds predominantly by electrostatic and hydrophobic forces to some residues in the BSA sub‐domains IIA and IIIA. Copyright © 2015 John Wiley & Sons, Ltd.     

Mechanism of protein extraction from the solid state by water-in-oil microemulsions

The extraction of solid-phase alpha-chymotrypsin, bovine serum albumin (BSA), and lysozyme by water-in-oil microemulsion (w/o-ME) solution containing Aerosol-OT (AOT) was thoroughly examined as a means to maximize protein solubilization in organic solvent media. Protein extraction occurred simultaneously with the adsorption of water and AOT by the solid protein. Water and AOT were desorbed at nearly equal rates, suggesting that both materials were desorbed together as micreomulsions. The solubilization of protein increased linearly with the ratio of solid protein to extractant solution except at a high value of the ratio, where most protein-containing microemulsions were desorbed. Based on our results, a mechanistic model was developed to describe the solid-phase extraction procedure. First, microemulsions are desorbed from solution by the solid protein, resulting in the formation of a solid protein-AOT-water aggregate. Second, when a protein in the solid phase binds to a sufficient number of microemulsions, the resulting aggregate's increased hydrophobicity drives its solubilization into lipophilic solvent. Third, through the exchange of materials between the solubilized precipitate and the remaining microemulsions, protein-containing w/o-MEs are formed. The presence of adsorption is further indicated by an isotherm existing between the water, AOT, and protein content of the resulting solid phase for each protein. The driving force behind adsorption is either AOT-protein interactions or the protein's affinity for microemulsion-encapsulated water, depending on the properties of the protein and the size of the microemulsions, in agreement with the model of P. L. Luisi [Chimia, 44: 270-282 (1990)]. The second step of our model is mass transfer limited for the extraction of solid alpha-chymotrypsin and BSA. The extraction of solid lysozyme was limited by the occurrence of an irreversible precipitation process. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 583-593, 1997.     

Stripping of nonionic surfactants from the coacervate phase of cloud point system for lipase separation by Winsor II microemulsion extraction with the direct addition of alcohols

Extractive microbial fermentation of lipase by Serratia marcescens ECU1010 in cloud point system was previously carried out in the cloud point system. The direct addition of different alcohols, including iso-butanol, 2-phenylethanol and 1-octanol, into the coacervate phase of the clear supernatant of the fermentation broth formed microemulsion, where the nonionic surfactants and lipase were unevenly partitioned between the different phases in the microemulsion system. The polarity of alcohols strongly affected the microemulsion type at room temperature condition. The results indicated that the Winsor II microemulsion, formed by the addition of iso-butanol or 2-phenylethanol as the organic solvent, favored the stripping of the nonionic surfactant into the O_m phase, whereas the lipase was left in the excess aqueous phase. However, the Winsor I microemulsion, formed by the addition of 1-octanol as the organic solvent, failed to separate the lipase from the nonionic surfactant in the coacervate phase of cloud point system, because the nonionic surfactant and lipase were partitioned into the W_m phase at the same time. Moreover, in the Winsor II microemulsion extraction with 2-phenylethanol as the organic solvent, in which case the protein–surfactant complexes were absent at the interface between the O_m phase and the excess aqueous phase, the high lipase recovery (above 80%) and good nonionic surfactant removal were achieved. The effect of nonionic surfactants on lipase activity was also presented.     

Superior Lithium Storage Capacity of α‐MnS Nanoparticles Embedded in S‐Doped Carbonaceous Mesoporous Frameworks

Herein, a Mn‐based metal–organic framework is used as a precursor to obtain well‐defined α‐MnS/S‐doped C microrod composites. Ultrasmall α‐MnS nanoparticles (3–5 nm) uniformly embedded in S‐doped carbonaceous mesoporous frameworks (α‐MnS/SCMFs) are obtained in a simple sulfidation reaction. As‐obtained α‐MnS/SCMFs shows outstanding lithium storage performance, with a specific capacity of 1383 mAh g^?1 in the 300th cycle or 1500 mAh g^?1 in the 120th cycle (at 200 mA g^?1) using copper or nickel foil as the current collector, respectively. The significant (pseudo)capacitive contribution and the stable composite structure of the electrodes result in impressive rate capabilities and outstanding long‐term cycling stability. Importantly, in situ X‐ray diffraction measurements studies on electrodes employing various metal foils/disks as current collector reveal the occurrence of the conversion reaction of CuS at (de)lithiation process when using copper foil as the current collector. This constitutes the first report of the reaction mechanism for α‐MnS, eventually forming metallic Mn and Li₂S. In situ dilatometry measurements demonstrate that the peculiar structure of α‐MnS/SCMFs effectively restrains the electrode volume variation upon repeated (dis)charge processes. Finally, α‐MnS/SCMFs electrodes present an impressive performance when coupled in a full cell with commercial LiMn_1/3Co_1/3Ni_1/3O₂ cathodes.     

Affinity extraction of BSA with reversed micellar system composed of unbound Cibacron Blue

Affinity Cibacron Blue 3GA (CB) dye in aqueous phase was directly transferred to the reversed micelles due to electrostatic interaction between anionic CB and cationic cetyltrimethylammonium bromide (CTAB). The bovine serum albumin (BSA) transfer to the reverse micelles increases significantly in a wide range of pH by the addition of a small amount of CB ( approximately 1.0-7.0% of the total surfactant concentration) to the aqueous phase. For pH < pI, the selectivity can be significantly improved with the presence of affinity CB because no BSA was extracted in the absence of CB. For backward extraction of BSA from the micellar phase with stripping aqueous solution, the addition of 2-propanol to the aqueous phase can recover almost all BSA (98.5%) extracted into the reverse micelles.     

油包水微乳液中抗体酶催化布洛芬酯选择性水解的酶学特性

根据过渡态理论设计和合成了能诱导产生催化选择性水解布洛芬甲酯的催化抗体的四面体硫酸盐半抗原,并与牛血清白蛋白(BSA)偶联制备成免疫源,通过免疫手段成功筛选出具有加速选择性水解生成S-布洛芬的特异性催化抗体.其Kcat,app/Kuncat,app达1.6x104.进一步地将催化抗体运用到W/O微乳体系(反胶束)中进行布洛芬酯的选择性水解研究,其动力学研究证明其催化过程同样遵循Michaelis.Menten方程.考察了pH值和温度对催化初速度影响,Wo(体系中水和琥珀酸二辛酯磺酸钠(AOT)的摩尔比)对催化初速度影响呈现为钟罩型,最适的Wo.为21.     

Extraction of bovine serum albumin with reverse micelles from glucosylammonium and lactosylammonium surfactants

Reverse micelle extraction is still in the stage of laboratory. Major limitation associated with use of synthetic surfactants in reverse micelle extraction process is the unfolding or denaturation of proteins. Sugar surfactants are thought non-toxic and environmentally benign, and can exhibit interesting interfacial properties, but the application of sugar-based surfactants in protein extraction is still limited. In the present study, we extracted bovine serum albumin (BSA) by using reverse micelles from glucosylammonium (GA) and lactosylammonium (LA) surfactants (with dicarboxylate as counter ion). It was found that under optimum condition, (1) the maximum forward extraction efficiency was ca. 86% with GA, while only around 50% with LA, and (2) almost all BSA solubilized in reverse micelles prepared from GA could be recovered into aqueous phase, while the recovery of BSA from the reverse micelles of LA was lower. In addition, the optimum extraction parameters were closely related to surfactant structure. Therefore, the electrostatic interaction, H-bonding and sugar head size should be important for BSA transfer.     

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。     

Protein‐based nanoformulations for α‐tocopherol encapsulation

Nanoparticles of BSA and silk fibroin (SF) with entrapped α‐tocopherol were produced via ultrasonic emulsification. Populations with particle size of 200–300 nm and highly negatively charged were obtained for all the tested formulations. Entrapment efficiencies of around 99% revealed the effective encapsulation of α‐tocopherol into the produced nanoformulations. Generally, these nanodevices did not induce significant cytotoxicity to human skin keratinocytes for all the concentrations tested. The developed formulations showed free radical scavenging of ABTS.⁺ ability resulting from the synergistic effect between the proteins in formulation and the entrapped tocopherol. Overall, the results contribute for the establishment of BSA:VO and BSA:SF:VO as biodegradable and non‐toxic nanoformulations for the functionalization of textile devices and controlled delivery of tocopherol into the skin.     

Study on the interaction between pelargonidin‐3‐O‐glucoside and bovine serum albumin using spectroscopic,transmission electron microscopy and molecular modeling techniques

The aim of this study is to evaluate the binding behavior between pelargonidin‐3‐O‐glucoside (P3G) and bovine serum albumin (BSA) using multi‐spectroscopic, transmission electron microscopy (TEM) and molecular docking methods under physiological conditions. Fluorescence spectroscopy and time‐resolved fluorescence showed that the fluorescence of BSA could be quenched remarkably by P3G via a static quenching mechanism, and there is a single class of binding site on BSA. In addition, the thermodynamic functions ΔH and ΔS were –21.69 kJ/mol and 24.46 J/mol/K, indicating that an electrostatic interaction was a main acting force. The distance between BSA and P3G was 2.74 nm according to Förster's theory, illustrating that energy transfer occurred. In addition, the secondary structure of BSA changed with a decrease in the α‐helix content from 66.2% to 64.0% as seen using synchronous fluorescence, UV/vis, circular dichroism and Fourier transform infrared spectroscopies, whereas TEM images showed that P3G led to BSA aggregation and fibrillation. Furthermore, site marker competitive experiments and molecular docking indicated that P3G could bind with subdomain IIA of BSA. The calculated results of the equilibrium fraction showed that the concentration of free P3G in plasma was high enough to be stored and transported from the circulatory system to its target sites to provide therapeutic effects. Copyright © 2015 John Wiley & Sons, Ltd.     

Sterilization mechanism of nitrogen gas plasma: induction of secondary structural change in protein

The mechanism of action on biomolecules of N₂ gas plasma, a novel sterilization technique, remains unclear. Here, the effect of N₂ gas plasma on protein structure was investigated. BSA, which was used as the model protein, was exposed to N₂ gas plasma generated by short‐time high voltage pulses from a static induction thyristor power supply. N₂ gas plasma‐treated BSA at 1.5 kilo pulses per second showed evidence of degradation and modification when assessed by Coomassie brilliant blue staining and ultraviolet spectroscopy at 280 nm. Fourier transform infrared spectroscopy analysis was used to determine the protein's secondary structure. When the amide I region was analyzed in the infrared spectra according to curve fitting and Fourier self‐deconvolution, N₂ gas plasma‐treated BSA showed increased α‐helix and decreased β‐turn content. Because heating decreased α‐helix and increased β‐sheet content, the structural changes induced by N₂ gas plasma‐treatment of BSA were not caused by high temperatures. Thus, the present results suggest that conformational changes induced by N₂ gas plasma are mediated by mechanisms distinct from heat denaturation.