反胶团萃取蛋白质的研究

本文以溶菌酶,胰蛋白酶和胃蛋白酶为对象,研究了水相pH值,离子强度、阳离子种类和蛋白质分子量等因素对反胶团萃取蛋白质的影响。结果表明,反胶团萃取的单级萃取率高,调节PH值和离子强度等工艺条件,就可以实现不同种类蛋白质的有效分离,可望成为一种生物产品分离的新方法。     

蛋白质的反胶团萃取机制及动力学模型研究进展

反胶团萃取是近年发展起来的分离和纯化生化物质的新方法,本文介绍了反胶团萃取蛋白质技术的原理和机制、影响反胶团中蛋白质稳定性的因素,改进的蛋白质反萃取工艺,反胶团的酶动力学研究以及反胶团萃取技术的研究展望。     

生物物质的分离新技术--反胶团萃取

反胶团萃取分离技术是一种新型的,有发展前途的生物产品分离技术。本文着重对反胶团的表面活性剂,各种影响因素(W0、pH、T等),动力学和热力学的理论研究以及目前的开发应用状况等多方面的现状进行综述,并对今后的发展进行展望。     

反胶团萃取蛋白质技术的反萃过程研究进展

反胶团萃取分离技术是一种新型的生物产品分离技术,本文简要介绍了反胶团萃取蛋白质技术的反萃过程的动力学,重点综述了在提高蛋白质反萃效率方面的研究进展,并对目前存在的问题、发展方向等进行了评述。     

TRPO-AOT 反胶团体系萃取牛血红蛋白的研究

反胶团是表面活性剂溶解在非极性溶剂中形成的、围绕一个“水核”的纳米级聚集体。液液反胶团萃取蛋白质技术,因对目标物质选择性好、容量大和能保持其活性而得到广泛研究^[1-9].在反胶团萃取蛋白质的研究中,多数作者采用单一表面活性剂AOT^[2]或季胺盐^[3]的反胶团体系。两种体系的共同弱点是:体系受离子强度、pH值等静电因素的影响大,直接影响萃取率,为了克服它们的不足,有人在AOT体系中加亲和试剂增强反胶团对蛋白质的亲和性^[4],加磷酸类阴离子表面活性剂^[5]、天然表面活性剂磷脂^[6]等以增强体系的萃取性能e人在季胺盐的反胶团体系中加非离子表面活性剂作助剂提高蛋白质的萃取率^[7],有人则反阴、阳和非离子表面活性剂混合形成反胶团提高某种酶的萃取容量^[8],本文用中性磷氧萃取剂三烷基氧膦（TRPO）与阴离子表面活性剂琥珀二辛酯磺酸钠（AOT）混合溶解在异辛烷中形成反胶团萃取牛血红蛋白（BHb）,比较AOT、TRPO及AOT三体系对牛血红蛋白（BHb）的萃取性能。     

氨基酸制备中的萃取技术

总结了氨基酸制备中的三大萃取技术:离子交换反应萃取,液膜分离萃取和反相胶团萃取及其在毛发水解氨基酸萃取分离方面的应用。     

猪心细胞色素C的反胶团萃取研究

本文研究了AOT/异辛烷对猪心CytC的萃取,分析了不同AOT浓度、pH值条件、体积比、阳离子种类以及离子浓度对反胶团萃取猪心CytC的影响,结果表明;AOT浓度增加,对CytC的萃取率提高;在pH6～9之间对CytC有较好的萃取效果;Mg~(2 )、Na~ 、K~ 、Ca~(2 )”、Mn~(2 )、Li~ 离子中以Li~ 和Mg~(2 )的效果最好,其顺序为:Li~ >Mg~(2 )>Na~ >Ca~(2 )>K~ ,而 Mg~(2 )在0.025mol/L时萃取率最好,可达96.5％;当油:水体积比增大时,萃取率也增大。     

反胶团萃取

反胶团萃取分离技术是一种新型的 ,有发展前途的生物产品分离技术。本文着重对反胶团的表面活性剂 ,各种影响因素 (W0 、pH、T等 ) ,动力学和热力学的理论研究以及目前的开发应用状况等多方面的现状进行综述 ,并对今后的发展进行展望     

包涵体纯化技术

20世纪90年代以来,基因重组技术得到很大的发展,基因工程产品的分离成本约占其全部成本的60%～80%,因此纯化技术越来越重要。着重介绍包涵体纯化技术,包括金属亲和层析,凝胶过滤层析,离子交换层析,疏水层析,双水相萃取技术和反胶团相转移技术近年来的进展情况。     

白地霉脂肪酶的双水相萃取和反胶团提取

对影响双水相萃取和反胶团提取脂肪酶的各种因素进行了探讨,并通过正交实验进一步优化提取条件,PEG浓度15%,(NH4)2SO4浓度22.5%,pH8.0的条件下进行双水相萃取,脂肪酶纯化倍数达到7.5倍;CTAB浓度150mmol/L,相体积比4/2,水相pH8.0,温度40℃的条件下进行反胶团提取,脂肪酶的比活力达到最大,但其比活力稍有下降,约为原来的0.9倍。     

Antimicrobial activity of AOT-isooctane reverse micelle as a bioseparation and biocatalysis tool

Abstract

The antimicrobial activity of different reverse micelles on microorganisms is been compared using the disc diffusion method. The bis (2-ethylhexyl) sodium sulfosuccinate (AOT) reverse micelle showed a more significant inhibitory effect than do other reverse. micelles, and it had an antimicrobial activity against a broad range of microorganisms. Results from an antimicrobial activity test of isooctane and a forward extraction containing soybean protein suggest that the surfactant was chiefly responsible for inhibiting microbes in AOT/isooctane reverse micelle, while isooctane hardly inhibited the microbial growth. The properties of S. aureus, cultured in the TSB with AOT reverse micellar solution, were identified by the SEM and SDS-PAGE fingerprinting of cell-wall proteins. It is concluded that the cell-wall of the S. aureus decreased in the TSB with AOT reverse micellar solution, and some cell protein subunits of the S. aureus did not occurr, especially between 14.4 and 42.7 kDa, while one new protein subunit at near 97.4 kDa occurred     

Impact of Surface Active Compounds on Iron Catalyzed Oxidation of Methyl Linolenate in AOT–Water–Hexadecane Systems

Edible oils contain minor surface active components that form micro-heterogeneous environments, such as reverse micelles, which can alter the rate and direction of chemical reactions. However, little is known about the role of these micro-heterogeneous environments on lipid oxidation of bulk oil. Our objective was to evaluate the ability of water, cumene hydroperoxide, oleic acid, and phosphatidylcholine to influence the structure of reverse micelles in a model oil system: sodium bis(2-ethylhexyl) sulfosuccinate (aerosol-OT; AOT) in n-hexadecane. The influence of reverse micelle structure on iron catalyzed lipid oxidation was determined using methyl linolenate as an oxidizable substrate. The size and shape of the reverse micelle were investigated by small-angle x-ray scattering, and water contents was determined by Karl Fischer titrations. Lipid hydroperoxides and thiobarbituric acid reactive substances were used to follow lipid oxidation. Our results showed that AOT formed spherical reverse micelles in hexadecane. The size of the reverse micelles increased with increased water or phosphatidylcholine concentration, but decreased upon addition of cumene hydroperoxide or oleic acid. Iron catalyzed oxidation of methyl linolenate in the reverse micelle system decreased with increasing water concentration. Addition of phosphatidylcholine into the reverse micelle systems decreased methyl linolenate oxidation compared to control and reverse micelles with added oleic acid. These results indicate that water, cumene hydroperoxide, oleic acid, and phosphatidylcholine can alter reverse micelle size and lipid oxidation rates. Understanding how these compounds influence reverse micelle structure and lipid oxidation rates could provide information on how to modify bulk oil systems to increase oxidative stability.     

Use of reverse micelles in membrane protein structural biology

Membrane protein structural biology is a rapidly developing field with fundamental importance for elucidating key biological and biophysical processes including signal transduction, intercellular communication, and cellular transport. In addition to the intrinsic interest in this area of research, structural studies of membrane proteins have direct significance on the development of therapeutics that impact human health in diverse and important ways. In this article we demonstrate the potential of investigating the structure of membrane proteins using the reverse micelle forming surfactant dioctyl sulfosuccinate (AOT) in application to the prototypical model ion channel gramicidin A. Reverse micelles are surfactant based nanoparticles which have been employed to investigate fundamental physical properties of biomolecules. The results of this solution NMR based study indicate that the AOT reverse micelle system is capable of refolding and stabilizing relatively high concentrations of the native conformation of gramicidin A. Importantly, pulsed-field-gradient NMR diffusion and NOESY experiments reveal stable gramicidin A homodimer interactions that bridge reverse micelle particles. The spectroscopic benefit of reverse micelle-membrane protein solubilization is also explored, and significant enhancement over commonly used micelle based mimetic systems is demonstrated. These results establish the effectiveness of reverse micelle based studies of membrane proteins, and illustrate that membrane proteins solubilized by reverse micelles are compatible with high resolution solution NMR techniques. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Extraction of flexibly structured protein in AOT reverse micelles: the flexible structure of protein is the dominant factor for its incorporation into reverse micelles

The extraction of flexibly-structured protein in Aerosol-OT (AOT)/isooctane reverse micelles was investigated. A flexibly-structured lysozyme was prepared by reduction and carboxymethylation of the disulfide bonds in the lysozyme molecule. For a comparison, lysozymes whose surface hydrophobicity was modified by monoacylation of the amino groups were also used. The extraction rate of the flexibly-structured lysozyme into the micellar phase was greater than that of the native and monoacylated lysozymes, although the free energy change of the lysozyme prepared by breaking the disulfide bonds was smaller than that of the lysozymes whose surfaces were monoacylated. Viscosity measurement of the micellar organic phase containing the modified lysozymes indicated that extraction of the flexibly-structured lysozyme changed the micelle–micelle interaction, while measurement of the interfacial tension between the AOT/isooctane and protein aqueous systems showed the flexibly-structured lysozyme to be the most amphiphilic in character. These results indicated that the flexible structure of a protein was more dominant than its surface hydrophobicity for its incorporation into reverse micelles, and that it leads to greater micelle–micelle interaction.     

Relaxation phenomena in systems of protein-containing reverse micelles of surfactants in organic solvents

The research was aimed to establish the equilibrium processes in protein-containing systems of AOT reverse micelles in octane. As chromophore label for tracing the kinetics of the process, the acid-base indicator, p-nitrophenol, was used. The establishing of the equilibrium in the reverse micelle system notably decelerated in the presence of a solubilized protein (native and stearoylated alpha-chymotrypsin). During the establishing of the equilibrium, the solubilized enzyme can be irreversibly inactivated. The level of the residual activity of the enzyme in the equilibrium system depended on the procedure of micellar system preparation. The methods have been offered to set up the equilibrium in the reverse micelle system without inactivation of the solubilized enzyme.     

Catalytic activity of lignin peroxidase and partition of veratryl alcohol in AOT/isooctane/toluene/water reverse micelles

The activity of lignin peroxidase (LiP) and the partition of its optimum substrate veratryl alcohol (VA) in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/toluene/water reverse micelles were studied in this paper to understand the microheterogeneous effect of the medium on the catalytic properties of LiP hosted in the reverse micelle. Results showed that LiP from Phanerochaete chrysosporium could express its activity in the reverse micelles, but its activity depended, to a great extent, on the composition of the reverse micelles. Optimum activity occurred at a molar ratio of water to AOT (ω₀) of 11, a pH value of 3.6, and a volume ratio of isooctane to toluene of 7–9. Under optimum conditions, the half-life of LiP was circa 12 h. The dependence of LiP activity on the volume fraction of water in the medium (θ), at a constant ω₀ value of 11, indicated that VA was mainly solubilized in the pseudophase of the reverse micelle. Based on the pseudobiphasic model and the corresponding kinetic method, a linear line can be obtained in a plot of apparent Michaelis constant of VA vs θ, and the partition coefficient of VA between the pseudophase and the organic solvent phase was determined to be 35.8, which was higher than that (22.3) between bulk water and the corresponding mixed organic solvent. H₂O₂ inhibited LiP at concentrations higher than 80 μM; this concentration value seems to be different from that in aqueous solution (about 3 mM). The differences mentioned above should be ascribed to the microheterogeneity and the interface of the AOT reverse micelle.     

An Alpha-Helical Peptide in AOT Micelles Prefers to be Localized at the Water/Headgroup Interface

A model alpha-helical peptide encapsulated in a reverse micelle is used to study the structure and dynamics of proteins under constrained environments that mimic the membrane-water environment in cells. Molecular dynamics simulations of the self assembly of systems composed of a peptide, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), water, and isooctane show that the peptide prefers to be located at the water/AOT headgroups interface. We explore the effect of the AOT headgroup charge and the peptide charge and find that the peptides migrate to the interface in all cases. These results show that the peptides prefer the constrained hydration environment of the AOT headgroups. The driving force for this configuration is the gain in entropy by released water molecules that otherwise would solvate the protein and surfactant headgroups     

反胶束中单宁酶的光学行为和稳定性

为了测定反胶束系统中单宁酶的光学行为和增溶方式,采用紫外分光光度法和荧光扫描技术对AOT水／异辛烷组成的反胶束体系中单宁酶和水相中单宁酶的光学行为进行研究,同时研究了不同反应体系中单宁酶的稳定性,并对单宁酶在反胶束体系中的增溶方式进行探讨。结果表明：反胶束体系与水相中的单宁酶,其光学行为存在很大差别。反胶束体系有利于单宁酶的稳定,脂肪醇作为反应底物,其碳链的增长有利于单宁酶在反胶束中的稳定性。单宁酶是以嵌入反胶束膜或与反胶束内膜接触的方式增溶的。     

1H-NMR of reverse micelles. II: Conformational studies of peptides and proteins in the AOT/water/isooctane system

The interaction of AOT reverse micelles with Met-enkephalin, the pancreatic secretory trypsin inhibitor (PSTI) and the epidermal growth factor (EGF) is examined by NMR methods and the three systems are compared. While Met-enkephalin adopts a folded conformation, PSTI appears to become highly flexible, suggestive of a non-specific interaction with the micelles. On the other hand, the EGF spectrum shows that, although the main globular features of the protein are retained in the presence of AOT, the C-terminal fragment has to rearrange its conformation when put in contact with the micelle wall.