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。     

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。     

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     

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。     

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的分离技术。本文着重对反胶团的各种影响因素(表面活性剂的种类、水与表面活性剂的摩尔比WO、pH值、温度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。     

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。     

用反胶束萃取法提取与分离蛋白质和酶

本文介绍了反胶束萃取的概念、体系、萃取原理及分配影响因素,综述了这一技术在蛋白质和酶的分离与纯化中的应用。     

从反胶束溶液中反萃牛血清白蛋白的研究

由近红外光谱证实,十六烷基三甲基溴化铵(CTAB)/正己醇-正辛烷反胶束溶液是牛血清白蛋白(BSA)增溶于非极性有机溶剂的较理想中介。研究了反萃液酸度、离子强度和种类等参数对BSA反萃率的影响,过低的溶液酸度导致BSA变性。适宜的反萃液为:1.0～2.0mol/L KBr,pH4.3～4.9。蛋白质的紫外光谱表明,BSA分子在料液,反胶束溶液和反萃液中的构象大致相同。此外,探讨了反胶束溶液循环使用的效率问题。最后,通过采用适当的相比,成功地实现了蛋白质的回收和浓缩。     

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。     

反胶束萃取技术分离胰激肽原酶

研究了用十六烷基三甲基溴化铵（CTAB）/正己醇/正辛烷反胶束溶液萃取和反萃取商业用胰激肽原酶时,水相pH值、离子强度和种类、CTAB浓度和助表面活性剂浓度等因素对分离效率的影响,并从反胶束微观结构给予解释。结果表明：［CTAB］＝0.02 mol•L-1,正己醇／正辛烷(V/V)＝1：5,萃取pH＝9.0,反萃pH＝7.0,萃取［KBr］＝0.1 mol•L-1,反萃［KBr］＝1.5 mol•L-1,反萃取加15％乙醇(V/V)时,萃取率接近100%,反萃取活性回收得率在80%以上。商业用酶的纯化倍数最高为1.97倍,粗酶为7.15倍,且粗酶纯化后比活在200U／mg以上,电泳分析证实了纯化效果,显示了很好的工业前景。     

Conformational transition and mass transfer in extraction of proteins by AOT–alcohol–isooctane reverse micellar systems

We examined quantitatively the effect of alcohols on protein and reverse micellar structure. We used circular dichroism (CD) to compare the effects of various alcohols on the protein structure, and percolation phenomena to evaluate the effects of various alcohols on reverse micellar structure. Upon the addition of alcohols to the bulk aqueous phase, proteins were denatured significantly, depending on the alcohol species and concentration, suggesting that use of alcohol directly to the stripping solution is not effective in back-extraction processes of proteins. In the present study, a new method, a small amount of alcohol is added to the surfactant–organic solution to improve the back-extraction behaviors of proteins. Practically, in the back-extraction process, the alcohols suppressing the cluster formation of reverse micelles (high value of β_t), remarkably improved the back-extraction behavior of proteins. In addition, the same alcohol molecules showed a positive effect on the rate and fraction of protein back-extraction. From a result of the CD measurement of the back-extracted proteins, it was known that the alcohols added to reverse micellar solution allowed the proteins to back-extract safely without causing structural changes. These results show that the values of β_t, defined by the variation of percolation processes, and the back-extraction behaviors of proteins have a good relationship, suggesting that the back-extraction processes were controlled by the micellar–micellar and protein–micellar interactions.     

Effect of operating variables on back-extraction characteristics of succinic acid from organic phase

Tri-n-octylamine (TOA) is an effective extractant for the recovery of succinic acid from fermentation broth. The recovery of succinic acid from organic phase depends on the operating variables such as temperature, pH, volume of aqueous phase, and use of displacer. In thermal recovery of succinic acid, 34% of succinic acid was recovered at 90°C. More than 90% of succinic acid was back-extracted by pH-swing. Efficiency of back-extraction was increased 23% as increasing volume of aqueous phase. Use of oleic acid as a displacer increased efficiency of back-extraction to 76%. Aqueous phase volume and concentration of oleic acid were controlled simultaneously to increase the efficiency of back-extraction and percent recovery of succinic acid reached to 90.     

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.     

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)).     

Enantioseparation of N-protected α-amino acid derivatives by liquid-liquid extraction technique employing stereoselective ion-pair formation with a carbamoylated quinine derivative

Two-phase liquid-liquid extraction experiments were undertaken to study the enantioselective transport of the chiral N-protected α-amino acid derivatives from an aqueous buffer solution into an organic phase employing highly lipophilic carbamoylated quinine as chiral selector and phase transfer carrier, respectively. The chiral separation, derived from enantioselective ion-pair formation and differential solubility in the aqueous and organic phases of diastereomeric associates thus formed has been shown to be primarily dependent on the structure of the selectand, the nature of the organic solvent, the molar ratio of a given chiral selector to selectand in the two phases, and the pH of the aqueous phase. Extracted enantiomers were recovered by back-extraction using a relatively polar acidic medium in which the selector is barely insoluble. Thus, the enantiomeric purity of N-(3,5-dinitrobenzoyl)-leucine exceeded 95% enantiomeric excess with 70% overall yield with a single extraction and back-extraction step. Chirality 9:268–273, 1997. © 1997 Wiley-Liss, Inc.     

Liquid-liquid extraction of lectin from Cratylia mollis seeds using reversed micelles

Extraction of lectin from seeds ofCratylia mollis, camaratu bean, with reversed micelles of 100 mM sodium di(2-ethylhexyl) sulfosuccinate/isooctane was performed firstly with affinity-purified lectin. The best conditions were extraction of the seed extract at pH 5 and back-extraction at pH 10, giving yields of 38% and 100%, respectively.     

On the use of organic extraction in the spin-trapping technique as applied to biological systems

A back-extraction methodology is presented which involves extraction of a spin adduct from an organic medium into an aqueous medium where its spectral parameters are well established. This technique should prove very useful in properly identifying spin adducts formed in organic media. Some of the hazards of extracting spin adducts into organic solvents for study are pointed out.     

Purification and spectroscopic properties of 124-kDa oat phytochrome

A simplified procedure for the isolation and purification of 124-kDa phytochrome from etiolated Avena seedlings has been developed using the method of ammonium sulfate back-extraction. After hydroxyapatite chromatography of seedling tissue extracts, the pooled phytochrome was subjected to ammonium sulfate back-extraction instead of the usual application to an Affi-Gel Blue column. The resulting phytochrome had specific absorbance ratios (SAR = A666/A280) ranging from 0.85 to 0.95. Subsequent Bio-Gel filtration chromatography yielded highly pure 124-kDa phytochrome with SAR values ranging from 0.99 to 1.13. The absorption maxima of 124-kDa phytochrome were at 280, 379, and 666 nm for the red absorbing form of phytochrome (Pr) and at 280, 400 and 730 nm for the far-red absorbing form (Pfr). The A730/A673 ratio in Pfr was found to be 1.5 to 1.6. The mole fraction of Pfr under red light photoequilibrium was 0.88. No dark reversion was detected within 5 h at 3 degrees C. A photoreversible far-uv-circular dichroism was observable with all phytochrome preparations examined. Fluorescence and phosphorescence lifetimes were measured to further characterize the differences between the phytochromes prepared under different conditions. The Trp fluorescence and phosphorescence lifetimes of Pr and Pfr with the chromophore "X", probably polyphenolic in nature, were significantly shorter than those of phytochrome without the contaminant X. The short lifetime of the fluorescence of the Pr chromophore is attributable to X in the former.     

Experimental investigation of citric acid reactive extraction with solvent recycling

Citric acid is one of the organic acids of which world market is growing every year. This paper proposes reactive extraction of citric acid with trioctylamine as an alternative to the classical method [1]. An experimental setup with solvent recycling is presented. As organic solvents were used: octanol, cyclohexanol, iso-butyl alcohol and paraffin oil. The removal efficiency is enhanced when the reactive extraction is accompanied by back-extraction using sodium carbonate as stripping agent.     

Sensitive gas chromatographic method for the determination of cinnarizine and flunarizine in biological samples

A sensitive method has been developed for the determination of the vasoactive compounds cinnarizine and flunarizine in plasma, urine and milk samples from man and animals. The procedure involves the extraction of the drugs and their internal standard from the biological samples at alkaline pH, back-extraction into sulphuric acid and re-extraction into the organic phase (heptane—isoamyl alcohol).The analyses were carried out by gas chromatography using a nitrogen-selective thermionic specific detector. The detection limit was 0.5 ng/ml of biological fluid and extraction recoveries were sufficiently high (87–94%).The method was applied to plasma samples from bioavailability studies of both cinnarizine and flunarizine in healthy volunteers, and to plasma, urine and milk samples from flunarizine-treated dogs.