Factors Affecting Trypsin Extraction by AOT Reversed Micelles and Observation by STM

In this article, the influence factors of trypsin extracted from crude pancreatin was investigated, and scanning turmeling microscope（STM） was used to observe the image of trypsin in butane-diacid-2-ethyl-hexyl-ester-sulfonic sodium （AOT）/iso-octane reversed micelles. The STM image showed that trypsins bounded in reversed micelles was rigid, which weakened its conjugative effect and caused maximum ultraviolet absorption and fluorescence emissive absorption moving toward blue waves. AOT concentration, pH and cations were the main influence factors of extraction. Specifically, extraction percentage of trypsin decreased with the increase of AOT concentration from 0.01 to 0.1mol·L^-1. When pH value is from 5.30 to 10.0, i.e. less than pI of trypsin, the extraction percentage is raised with the different increase of pI-pH, but when the pH value is less than 5.20, the extraction percentage is decreased with the acidity added. Besides, the extraction efficiency is negative, related with the concentrations of Ca^2＋, Na^＋, K^＋ which were in the range of 0.2-1.0mol.L^-1, and influence of concentration of Ca^2＋ is greater than that of Na^＋, and K^＋ which has the minimum impact with the same concentration. Finally, optimum conditions to extract trypsin were： AOT reversed micelles 0.05mol·L^-1, trypsin concentration in crude pancreatin solution 3mg·ml^-1, pH 5.2-- 5.3, ratio （by volume） of extraction phase to strip-extraction phase 1 ： 1, and time of 5min. The corresponding percentage of extraction was 22.7% and specific activity was 78.9 N-benzoyl-L-arginlne ethyl ester （BAEE） U·mg^-1 protein, three times than that in crude pancreatin. There was no lipase and amylopsin activity was decreased to 1/5 of crude pancreatin. Partly purifying solution was treated by condition mentioned above with 0.05mol·L^-1 ceryl-trimethyl-ammonium bromide （CTAB）, total extraction percentage of trypsin was 74.18% and specific activity was 3148.3 BAEE U·mg^-1, i.e. 48.16 times purer than that in crude pancreatin. Through sodium dodecyl sulfate-polyacryl amide gel electrophoresis （SDS-PAGE） and image analysis of extracted product, there were only three bands in the trypsin, while seven in crude pancreatin, and electrophoresis location of main bend was almost identical with the standard enzyme.

Enzymatic reaction of ethanol and oleic acid by lipase and lignin peroxidase in rhamnolipid(RL) reversed micelles

An environment friendly bio-surfactant of rhamnolipid(RL) was used as a solvent. The enzymatic reaction of oleic acid catalyzed by lipase and lignin peroxidase(lip) was evaluated. The optimum conditions of enzymatic reaction catalyzed by lipase(lip) were water to amphiphile molar ratio of 30(20), RL of 60(60) critical micelle concentration(CMC), pH of 7.0(3.0) and temperature of 40(30) °C, respectively. The change of enzyme conformation indicates that, for catalytic of lipase, water content is the most important factor of the enzymatic reaction of oleic acid, and p H for lip. With individual optimum conditions, the enzymatic efficiency of oleic acid catalyzed by lipase is higher than that by lip. In the presence of ethanol, the enzymatic reaction of oleic acid catalyzed by lipase suits Ping-Pong Bi-Bi mechanism. As an alternative to chemical reversed micelles, the RL reversed micelles are promising methods to enzymatic reaction of oleic acid.

Mass transfer process of peanut protein extracted by bis(2-ethylhexyl)sodium sulfosuccinate reverse micelles

The liquid-liquid extraction method using reverse micelles can simultaneously extract lipid and protein of oilseeds,which have become increasingly popular in recent years.However,there are few studies on mass transfer processes and models,which are helpful to better control the extraction process of oils and proteins.In this paper,mass transfer process of peanut protein extracted by bis(2-ethylhexyl)sodium sulfosuccinate(AOT)/isooctane reverse micelles was investigated.The effects of stirring speed(0,70,140,and 210 r/min),temperature of extraction(30,35,40,45,and 50℃),peanut flour particle size(0.355,0.450,0.600,and 0.900 mm)and solidliquid ratio(0.010,0.0125,0.015,0.0175,and 0.020 g/mL)on extraction rate were examined.The results showed that extraction rate increased with temperature rising,particle size reduction as well as solid-liquid ratio increase respectively,while little effect of stirring speed(P>0.05)was observed.The apparent activation energy of extraction process was calculated as 10.02 kJ/mol and Arrhenius constant(A)was 1.91 by Arrhenius equation.There was a linear relationship between reaction rate constant and the square of the inverse of initial particle radius(1/r_(0)^(2))(P<0.05).This phenomenon and this shrinking core model were anastomosed.In brief,the extraction process was controlled by the diffusion of protein from the virgin zone interface of particle through the reacted zone and it was in line with the first order reaction.Mass transfer kinetics of peanut protein extracted by reverse micelles was established and it was verified by experimental results.The results provide an important theoretical guidance for industrial production of peanut protein separation and purification.

TR-ESR Investigation on Reaction of Vitamin C with Excited Triplet of 9,10-phenanthrenequinone in Reversed Micelle Solutions

Time-resolved electron spin resonance has been used to study quenching reactions between the antioxidant Vitamin C （VC） and the triplet excited states of 9,10-phenanthrenequinone （PAQ） in ethylene glycol-water （EG-H2O） homogeneous and inhomogeneous reversed micelle solutions. Reversed micelle solutions were used to be the models of physiological environment of biological cell and tissue. In PAQ/EG-H2O homogeneous solution, the excited triplet of PAQ （3PAQ＊） abstracts hydrogen atom from solvent EG. In PAQ/VC/EG-H2O solution, 3pAQ＊ abstracts hydrogen atom not only from solvent EG but also from VC. The quenching rate constant of 3pAQ＊ by VC is close to the diffusion-controlled value of 1.41 × 108 L/（mol.s）. In hexadecyltrimethylammonium bromide （CTAB）/EG-H2O and aerosol OT （AOT）/EG- H2O reversed micelle solutions, 3pAQ＊ and VC react around the water-oil interface of the reversed micelle. Exit of 3pAQ＊ from the lipid phase slows down the quenching reaction. For Triton X-100 （TX-100）/EG-H2O reversed micelle solution, PAQ and VC coexist inside the hydrophilic polyethylene glycol core, and the quenching rate constant of 3pAQ＊ by VC is larger than those in AOT/EG-H2O and CTAB/EG-H2O reversed micelle solutions, even a little larger than that in EG-H2O homogeneous solution. The strong emissive chemically induced dynamic electron polarization of As＇- resulted from the effective TM spin polarization transfer in hydrogen abstraction of 3pAQ＊ from VC.

Selective Affinity Separation of Yeast Alcohol Dehydrogenase by Reverse Micelles with Unbound Triazine Dye

The reversed micelles were formed with cationic cetyltrimethylammonium bromide (CTAB) as surfactant and n-hexanol as cosolvent in the CTAB (50mmol.L-1)/hexanol (15% by volume)/hexane system. Cibacron Blue 3GA (CB) as an affinity ligand in the aqueous phase was directly introduced to the reversed micelles with electrostatic interaction between anionic CB and cationic surfactant. High molecular weight (Mr) protein, yeast alcohol dehydrogenase (YADH, Mr = 141000) from baker's yeast, has been purified using the affinity reversed micelles by the phase transfer method. Various parameters, such as CB concentration, pH and ionic strength, on YADH forward and backward transfer were studied. YADH can be transferred into and out from the reversed micelles under mild conditions (only by regulation of solution pH and salt concentration) with the successful recovery of most YADH activity. Both forward and backward extractions occurred when the aqueous phase pH＞pI with electrostatic attraction between YADH and CTAB. The recovery of YADH activity and purification factor have been improved with addition of a small amount of affinity CB. The recovery of YADH activity obtained was ～99% and the purification factor was about 4.0-fold after one cycle of full forward and backward extraction. The low ionic strength in the initial aqueous phase might be responsible for the YADH transfer into the reversed micellar phase.

New Development of Reverse Micelles and Applications in Protein Separation and Refolding

Reverse micelles bring mild and effective microenvironments in organic solvent that contain bitmolecules, which have attracted immense attention for application in the isolation of proteins, protein refolding, and enzymatic reaction. In this review, the application of reverse micelles for protein separation and refolding has been briefly summarized and various reverse micellar systems composed of different surfactants, including ionic, non- ionic, mixed, and affinity-based reverse micelles, have been highlighted. It illustrates especially the potential application of the novel affinity-based reverse micelles consisting of biocompatible surfactant coupled with affinity ligands. Moreover, the importance to develop universal affinity-based reverse micelles for protein separation and refolding in the downstream processing of biotechnology has been pointed out.

Activity Regulation of Lignin Peroxidase from Phanerochaete chrysosporium in Nonionic Reversed Micellar Medium

The activity of lignin peroxidase (LiP) in reversed micelles of polyoxyethylene lauryl ether (Brij30) changed with the molar ratio of water to the surfactant and the denaturant concentration of guanidinium chloride. At low water contents the activity of LiP could be enhanced by the denaturant at moderate concentration. This phenomenon, together with the spectral characteristics of the intrinsic fluorescence of LiP, suggested that the conformation of the active center of LiP was flexible.

Mechanisms of Cytochrome C Extraction by Reverse Micelles

The extraction of cytochrome C was carried out by means of phase transfer technique with three different reverse micellar systems, i.e. , a CTAB micellar solution in n butyl alcohol chloroform(volume ratio 4∶1), an AOT micellar solution in isooctane and a SDSS D 2EHPA micellar solution in isooctane. The extraction mechanisms were studied. The results show that the extraction mechanisms for the same proteins with different types of reverse micellar systems can be distinct. The extraction of cytochrome C with CTAB and SDSS D 2EHPA reverse micellar systems are carried out according to the mechanism of electrostatic interaction. However, in the extraction of cytochrome C with the AOT reverse micellar system, the electrostatic interaction between the protein and the surfactant is not important.

Size Changes of Reverse Micelles after Extraction of Peanut Protein and Their Forward Extraction Rates

The aim of this study was to detect the size changes of reverse micelles after extraction of peanut protein and their forward extraction rates. Factors that affect the size of reverse micelles and the extraction of peanut protein were also investigated. The size of reverse micelles and the size changes were measured according to the theory of dynamic light scattering under different conditions such as different sodium bis(2-ethylhexyl) sulfosuccinate(AOT) concentrations, p H values, ion concentrations, and salt species.With the increase of AOT surfactant concentrations in a certain range, the size of empty and full reverse micelles increased and the forward extraction rate decreased. The effect of pH on empty reverse micelles was not significant. However, the effect of pH on the full reverse micelle size and forward extraction rate were significant. Its forward extraction rate increased to the maximum39.6% at pH 7.5. The increase of the salt concentration of a buffer solution in a certain range decreased the size of empty and full reverse micelles and reduced the forward extraction rate of peanut protein. Ionic species had important effects on reverse micelles and peanut protein extraction. An increase in the amount of buffer solution enlarged the empty reverse micelle size in 0.03%-0.11%(V/V). However, it did not translate to a larger reverse micelle size. The size of the empty reverse micelles containing K_2SO_2 reached 24.1 nm with a 0.19%(V/V) buffer solution added. The sizes of the full reverse micelles were larger than those of the empty reverse micelles after forward extraction. However, maximum sizes were achieved with the addition of a 0.03%(V/V) buffer solution. The amount of 0.03%(V/V) buffer solution added was appropriate for extracting peanut protein.

Polymerization of o-Phenylenediamine Catalyzed byHemeproteins Encapsulated in Reversed Micelle

Hemeproteins encapsulated in reversed micelle formulated with di-2-ethylhexyl sulfosuccinate(AOT) was found to catalyze the polymerization of o-phenylenediamine(o-PDA) with hydrogen peroxide, whereas o-PDA catalyzed by hemeproteins dissolved in water could only form its trimers. As the nanostructural environment in reversed micelle acts as a certain orientation surrounding medium, it offers a strong electrostatic field that alters the reductive potential of Fe 3+/Fe 2+(E m7) in the heme of hemeproteins and thus increases the catalytic activity of peroxidase accordingly. According to the results of UV-Vis, 1H NMR and FTIR, the polymer catalyzed by hemoglobin(Hb) in reversed micelle was presumed to be constructed of lines and trapeziforms alternatively.

Absorption Complex between Porphyrin and Phenothiazine in Reverse Micelles

The interaction between amphiphilic porphyrin and phenothiazine in AOT/isooctane/water reverse micelle was investigated by UV-Vis spectra. A new absorption complex between the two species is formed in such circumstances, which is ascribed to the enrichment of the components by the reverse micelle. The fluorescence quenching of CHTTP by PTH becomes more efficient after the formation of the absorption complex.

Porphyrins in Reverse Micelles: the Side-chain Length and the Triplet-state Lifetime

Using bis(2-ethylhexyl) sodium sulfosuccinate (AOT) as surfactant, two amphiphilic porphyrin terminated with imidazole were studied in AOT/iso-octane/water reverse micelles, intending to mimic the relationship between microenvironments in organism and the amphiphilic properties of porphyrins for photodynamic therapy drugs.

Analysis on the Characteristic Properties of PEDOT Nano-particle Based on Reversed Micelle Method

Based on the study of a new type of conducting polymer poly （3,4-ethylenedioxythiophene） （PEDOT）,we focussed on the preparation and characteristics of PEDOT nanoparticles made by reversed micelle method.Moreover,we deeply investigated the optical,electrical and the thermal stability of PEDOT nanoparticles.The main results are as follows： the small-sized PEDOT nanoparticles were prepared and utilized by different methods,such as ultraviolet/visible （UV-Vis） spectroscopy,Fourier-transform infrared （FT- IR） spectrum,scanning electron microscopy（SEM） and so on.The results show that the amount of oxidizer,ultrasonic treatment,polymerizing temperature and doping degree can influent morphology,electrical ability and gas sensitivity of PEDOT nanoparticles.The Bragg peaks of nanoparticles at 6.7°,12.7°,25° were observed by XRD and the better orientation of molecular chain was attributed to the effective doping of toluene-p-sulfonic acid,which also resulted in an enhancement of thermal stability of nanoparticles than conventional PEDOT.

Terahertz spectroscopy of water in nonionic reverse micelles

The dynamics of water within a nanopool of a reverse micelle is heavily affected by the amphiphilic interface.In this work,the terahertz(THz)spectra of cyclohexane/Igepal/water nonionic reverse micelle mixture are measured by THz timedomain spectroscopy and analyzed with two Debye models and complex permittivity of background with volume ratios.Based on the fitted parameters of bulk and fast water,the molar concentration of all kinds of water molecules and hydration water molecule number per Igepal molecule are calculated.We find that slow hydration water has the highest proportion in water when the radius parameterω_(0)<10,while bulk water becomes the main component whenω_(0)≥10.The feature radius ratio of nonhydrated and hydrated water to total water nanopool is roughly obtained from 0.39 to 0.85 with increasingω_(0).

Phase separation in solvent extraction of cobalt from acidic sulfate solution using synergistic mixture containing dinonylnaphthalene sulfonic acid and 2-ethylhexyl 4-pyridinecarboxylate ester

Phase separation rate is a critical character in determining the usefulness of a solvent extraction system in hydrometallurgy. A survey of the synergistic mixture containing dinonylnaphthalene sulfonic acid (HDNNS) and 2-ethylhexly 4-pyridinecarboxylate ester (4PC) for the extraction of cobalt from acidic single metal sulfate solution was carried out to suggest how the physicochemical properties and the morphology of the reverse micelles in the loaded organic phase affect the phase separation. The results show that effective parameters affecting the phase separation are the viscosity and the excess water uptake of the loaded organic phase. It is obvious that the specific settling rate (SSR) decreases with the apparent increase of these two parameters. The measurement of small angle X-ray scattering (SAXS) proves that the morphology of the reversed micelles in the loaded organic phase changes evidently with the change of the specific settling rate (SSR).

Determination of Teicoplanin A2's Partition Coefficient in Different Liquid-Liquid Extraction Systems

Teicoplanin is one of the macrocyclic glycopeptide antibiotics, which is active against Gram-positive bacteria, and has attracted a lot of attention in the field of chiral separation recently. In this work, the partition coefficients and extraction ratio of teicoplanin in three different solvent systems were studied: conventional extraction, reactive extraction and reverse micelle extraction. With conventional solvent extraction, n-butanol demonstrated high partition coefficient for teicoplanin, but low extraction ratio because of its high solubility in water. Reactive extraction of teicoplanin showed the highest partition coefficient with almost 100% recovery in organic phase when tri-n-octylmethyl ammonium chloride (TOMAC) was used as extractant and pH value was above 5.0. A reverse micelle system, consisted of isooctane +10mmol-L-1 TOMAC +1% n-octanol, also offered high separation factor for teicoplanin. The results are beneficial for the design of teicoplanin separation and purification process.

Effects of postthermal treatment and UV irradiation on the structure of titania-polyacrylate nanocomposites

The effects of postthermal treatment and irradiation time on the structure and thermal stability of TiO2/polyacrylate nanocomposites by a sol-gel process in reverse micelles and subsequent rapid photopolymerization were investigated, and the hybrid films were characterized by thermal gravimetry analysis （TGA）, X-ray photoelectron spectrum （XPS）, and atomic force microscopy （AFM）. XPS data suggested that the prolongation of irradiation time and the postthermal treatment promoted titania formation, with the former affecting more remarkably. TGA data showed that TiO2-hybrid films could upgrade the decomposition onset temperature （Tonset） as well as the temperature at which there is a maximum mass loss rate （Tmax）. AFM data demonstrated that the inorganic titania particles with a mean diameter of 25.26-28.84 nm were homogeneously distributed in the organic matrix.

Low-temperature Growth of Single-crystal SrCO_3 Nanoneedles

Single-crystal SrCO3 nanoneedles were synthesized in reverse micelles at low temperature. The products were characterized by X-ray diffraction, X-ray energy dispersive spectrometer, transmission electron microscopy and selected area electronic diffraction. The influences of experimental conditions on the morphologies of the products were discussed. The growth mechanism of SrCO3 nanoneedles in reverse micelles were proposed.

Effect of Dissolved CO_2 on Protein Solubilization in Reverse Micelle

Effect of dissolved CO2 on bovine serum albumin (BSA) solubilization in the reverse micelles of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) in 2,2,4-trimethylpentane (iso-octane) has been studied at 308.2 K. It was found that BSA can be precipitated completely by CO2 while the AOT and water remain in the iso-octane continuous phase. This opens up a possibility for recovery of pure protein solids directly from reverse micellar solutions.