Study on PEG-（NH4）2SO4 Aqueous Two-Phase System and Distribution Behavior of Drugs

The distribution behavior of chlorpromazine hydrochloride (CPZ), procaine hydrochloride (PCN) and procaine amide hydrochloride (PCNA) in polyethylene glycol (PEG800 or PEG1500)-(NH4)2SO4 aqueous two-phase systems has been investigated. The result shows that the PEG-(NH4)2SO4 aqueous two-phase system has potential extraction capability in small molecular drug separation. In PEG800-(NH4)2SO4 system, the extraction efficiencies (E) of CPZ, PCN and PCNA amount to 92.8%, 74.5% and 74.4%, respectively, with the distribution coefficients (KD) being 25.7, 5.9 and 5.8, correspondingly. In PEG1500-(NH4)2SO4 system, the extraction efficiencies (E) of CPZ,PCN and PCNA are 93.7%, 71.3% and 63.2%, respectively, with distribution coefficients (KD) of 39.6, 6.6 and 5.0,correspondingly. Based on the study on ultraviolet and fluorescence spectra and also distribution behavior of the drugs in PEG-(NH4)2SO4 aqueous two-phase system, extraction mechanism was further proposed that both hydrogen bond and hydrophobic interaction are involved in extraction.     

聚乙二醇双水相萃取光度法测定镍

用水溶性高聚物、表面活性剂及有机物与无机盐形成的双水相体系萃取分离色素、蛋白质及测定金属离子已有报道[1-5].     

双水相萃取结合液相色谱法分离蛋白质

建立了PEG/( NH4)2SO4双水相体系萃取富集,结合液相色谱分离分析多种蛋白质的方法.考察了无机盐种类和浓度、PEG分子量、pH值和温度等因素对双水相形成以及对细胞色素C、肌红蛋白、牛血清白蛋白、溶菌酶、胰蛋白酶分配行为的影响.结果表明,上述5种蛋白在室温、pH 3.5～9.0范围内,可在15％ PEG-4000/10％ (NH4)2SO4双水相体系中得到富集,且主要集中在下相.同样条件下,血清中的高丰度蛋白在上下相均有分配,下相分配量较大.通过双水相萃取分离蛋白质及对液相色谱一定时间段的色谱峰收集,可初步实现血清中高丰度蛋白质的分离去除.     

聚乙二醇双水相萃取光度法测定铼

研究了聚乙二醇双水相体系中,金属离子络合物及萃取剂的光谱行为,研究了酸度、缓冲溶液用量、盐用量、萃取剂用量及表面活性剂对萃取铼的影响,并通过控制一定条件,可实现了混合离子的分离。     

Purification of glucose-6-phosphate dehydrogenase from baker’s yeast in aqueous two-phase systems with free triazine dyes as affinity ligands

To improve the selectivity of glucose-6-phosphate dehydrogenase (G6PDH) extraction by an aqueous two-phase system, a simple and inexpensive affinity aqueous two-phase system using unbound reactive triazine dyes as ligands was introduced. In a polyethylene glycol (PEG)/hydroxypropyl starch (PES) system, the unbound free triazine dyes, Cibacron Blue F3GA and Procion Red HE3B, partitioned unevenly in the top PEG-rich phase and thus showed an affinity effect on G6PDH, but no influence on hexokinase. The various parameters investigated were pH of the system, buffers, molecular weight of PEG, and ligand type and concentration. A two-step affinity extraction process was established for the purification of G6PDH from baker’s yeast. The total yield of G6PDH was 66.9% and purification factor was 2.35.     

组合表面活性剂-盐-水双水相体系萃取水杨酸和洛美沙星

通过实验选择聚乙二醇(PEG1500)与聚乙烯吡咯烷酮(PVP30000)组合表面活性剂(NH4)2SO4-H2O形成双水相体系及其成相条件。研究水杨酸(APC)和洛美沙星(LFXC)氢键型药物在该双水相体系中的分配行为。结果表明：该体系对APC和LFXC的萃取率分别为102％和69．0％。通过药物在该体系中的紫外吸收光谱及分子荧光光谱的变化,验证其萃取药物的机理。     

聚乙二醇-硫酸铵双水相萃取次甲基蓝的研究

利用分光光度法研究了次甲基蓝在聚乙二醇-硫酸铵双水相体系的萃取行为,探索了质量配比(mPEc/m(NH4)2SO4)、温度和次甲基蓝的浓度对双水相及次甲基蓝萃取率(Y)的影响.实验表明:(1)在一定的温度和浓度下,随着mPEC/m(NH4)2SO4的减小,分配系数K( cup/clow)增加,萃取率Y略有减小;(2)在一定的质量配比和浓度下,分配系数K(cup/clow)随温度的升高而增大,但萃取率Y却基本保持不变;(3)在一定的质量配比和温度下,随着次甲基蓝浓度的增加,分配系数K(cup/clow)增大;萃取率Y略有增加.在mPEG/m( NH4) 2SO4为1.50∶1,c次甲基蓝=8.915×10-5mol/L、t=30℃的实验条件下,K(cup/clow)=22.14,萃取率(y)可达95.43％.     

Partition behavior and partial purification of hexokinase in aqueous two-phase polyethylene glycol/citrate systems

This study dealt with the partition behavior and partial purification of hexokinase (HK) from baker’s yeast by liquid-liquid extraction using aqueous two-phase polyethylene glycol (PEG)/citrate systems. First, we investigated the effect of agitation type (vortex and 8 rpm rotation) on the stability of the system, and then the effects of sodium citrate concentration, PEG concentration, and molar mass of PEG on the partition coefficient of this enzyme by using a 2⁵ factorial experimental design. The results of this factorial experiment showed the possibility of a partial purification of HK by using two extraction steps, since the enzyme preferentially migrated to the top phase and the total proteins (mainly contaminants) remained in the bottom phase. The purification factor (Pur _TOP) of the enzyme in the top phase was 1.87, and the partition coefficient of the total proteins (K _Prot ) was 0.47.     

Partitioning of Porcine Pancreatic Lipase in a Two-Phase Systems of Polyethylene Glycol/Potassium Phosphate Aqueous

The hydrolysis of triglycerides at the oil–water interface, synthesis of esters and transesterification in microaqueous conditions are catalysed by lipase. For its application, a proper purification method was necessary. This study examined the application of an aqueous two-phase system to partition porcine pancreatic lipase. The influence of molecular weight and concentration of polyethylene glycol (PEG), tie line length (TLL), potassium phosphate concentration, sodium chloride (NaCl) addition and temperature in the partition was studied. The enzyme was more efficiently purified in PEG 8,000 at 14.5 °C (PF?=?3.89-fold), presenting more recoveries at the top phase with shorter TLL and lower concentrations of PEG and potassium phosphate. Moreover, the increase of these variables repressed the purification and the further addition of NaCl did not promote the purification of the enzyme. These results demonstrated the efficiency of the aqueous two-phase system on lipase purification.     

Integrated Process Production and Extraction of the Fibrinolytic Protease from Bacillus sp. UFPEDA 485

Fibrinolytic proteases are enzymes that degrade fibrin; these enzymes are a promising alternative for thrombolytic therapy, and microorganisms produce them. The aim of this study was to evaluate the optimum conditions for the integrated production and purification of fibrinolytic protease from Bacillus sp. UFPEDA 485. Extractive fermentation was carried out in a culture medium containing soybean flour and by adding polyethylene glycol (PEG) and Na₂SO₄ according to a 2³ experimental design. In all assays, the enzyme preferentially partitioned to the bottom phase (K?<?1), with an optimum activity of 835 U ml^?1 in the bottom phase (salt-rich phase). The best conditions for extractive fermentation were obtained with 18 % PEG 8000 and 13 % Na₂SO₄. Characterization showed that it is a metalloprotease, as a strong inhibition—residual activity of 3.13 %—occurred in the presence of ethylenediaminetetraacetic acid. It was also observed that enzymatic activity was stimulated in the presence of ions: CaCl₂ (440 %), MgCl₂ (440 %), FeSO₄ (268 %), and KCl (268 %). The obtained results indicate that the use of a low-cost substrate and the integration of fermentation with an aqueous two-phase system extraction may be an interesting alternative for the production of fibrinolytic protease.     

One-step purification of alpha-amylase from the cultivation supernatant of recombinant bacillus subtilis by high-speed counter-current chromatography with aqueous polymer two-phase systems

Purification of alpha-amylase from the cultivation supernatant of recombinant Bacillus subtilis by high-speed counter-current chromatography (HSCCC) in polyethylene glycol (PEG) 4000-inorganic salt aqueous polymer two-phase systems was studied. The effects of sodium chloride concentration on the partition coefficients of alpha-amylase and total protein were respectively tested in PEG4000-phosphate and PEG4000-citrate aqueous polymer two-phase systems to find the proper range of sodium chloride concentration for the HSCCC purification of alpha-amylase. Alpha-amylase was purified from the cultivation supernatant by HSCCC in PEG4000-phosphate system containing 2% (w/w) sodium chloride, yet with considerable loss of activity. PEG4000-citrate aqueous polymer two-phase system containing 2% (w/w) sodium chloride and supplemented with 0.56% (w/w) CaCl2 as protective agent was then successfully applied to purify alpha-amylase from cultivation supernatant by HSCCC to homogeneity and significantly increased the recovery of alpha-amylase activity from around 30 to 73.1%.     

醇与离子液体二元双水相体系萃取盐酸多西环素

基于小分子醇双水相体系和离子液体双水相体系,建立了正丙醇与亲水性离子液体1-丁基-3-甲基咪唑四氟硼酸[Bmim]BF4和(NH4)2SO4形成的二元双水相体系萃取盐酸多西环素的新方法。考察了(NH4)2SO4含量、正丙醇用量、pH值、离子液体含量以及盐酸多西环素含量对盐酸多西环素分配行为的影响。结果表明:当醇和离子液体二元双水相体系的pH值在4.0～5.0范围内,(NH4)2SO4含量为34%,且盐酸多西环素的质量浓度在25～95 mg/L之间时,该体系对盐酸多西环素的萃取率可达90.26%～95.71%,分配系数可达62.452～149.401。     

浊点萃取-异辛烷反萃取-气相色谱测定茶叶中拟除虫菊酯农药残留

建立了浊点萃取-异辛烷反萃取-气相色谱(ECD)检测茶叶中联苯菊酯(Bifenthrin)、甲氰菊酯(Fenpropathrin)、功夫菊酯(Cyhalothrin)、氯菊酯(Permethrin)、氰戊菊酯(Fenvalerate)、溴氰菊酯(Deltamethrin)6种拟除虫菊酯农药残留的方法.对含1.2%(m/V)聚乙二醇6000(PEG6000)表面活性剂和40%(m/V)(NH4)2SO4的6种拟除虫菊酯溶液进行加热萃取,所获得的富集相用异辛烷超声反萃取,并经离心对上层异辛烷溶液进行进一步净化处理,即可获得富集倍数达75倍的6种农药.本方法的检出限(LOD)为:联苯菊酯、甲氰菊酯和功夫菊酯0.4 μg/kg;氰戊菊酯2.1 μg/kg;氯菊酯和溴氰菊酯3.0 μg/kg;用本方法测定了新鲜茶叶中6种拟除虫菊酯农药,含量分别为4.17, 4.15, 4.09, 4.01, 3.93和3.51 μg/kg.在上述茶叶样品中添加20 μg/kg 的6种农药后测定,添加回收率为72.3%～85.6%; 相对标准偏差为2.2%～5.6%.     

成相无机盐种类对PEG双水相相平衡影响

不同种类无机盐分别与PEG1000、PEG2000、PEG,4000和PEG6000构成双水相体系,在298.15K下,采用浊点滴定法研究双水相体系相平衡,通过Merchuk经验方程拟合实验点确定双水相体系的双节线,进而考察成相无机盐种类对PEG双水相相平衡的影响.结果表明:具有相同阴离子,易于形成双水相的无机盐顺序为...     

System Establishment of ATPS for One-Step Purification of Glutamate Decarboxylase from <Emphasis Type="Italic">E. coli</Emphasis> After Cell Disruption

The partition of glutamate decarboxylase (GAD) from Escherichia coli in polyethylene glycol (PEG) and sodium sulfate aqueous two-phase systems (ATPS) has been explored with the purpose of establishing a phase system for the purification of GAD after cell disruption. The results showed that the partitioning of GAD was slightly influenced by PEG molecular weight (MW) but depended on the tie line length (TLL) and NaCl and loading sample concentrations. The optimum system obtained for GAD purification was composed of a PEG MW of 4,000, TLL of 63.5%, a volume ratio of 2.31, a loading sample concentration of 0.4 g/mL, which produced a GAD recovery of 90% with the purification fold of 73. Furthermore, the feasibility of directly purifying GAD from the cell disrupts using ATPS was evaluated. The established ATPS for GAD purification exhibited an efficient integrated purification process compared to the reported purification process in terms of purification efficiency and recovery.     

Purification of phospholipase D by two-phase affinity extraction

An aqueous two-phase system of polyethylene glycol (PEG)-salt was used for purification of phospholipase D (PLD) from peanuts and carrots. Alginate, a known macroaffinity ligand for PLD, was incorporated in the PEG phase and resulted in 91 and 93% of the enzyme activity (from peanuts and carrots, respectively) getting partitioned in the PEG phase. The elution of the enzyme from alginate was facilitated by exploiting the fact that the latter can be reversibly precipitated in the presence of Ca2+. The enzyme was eluted from the polymer by using 0.5 M NaCl. Peanuts and carrots PLD could be purified 78- and 17-fold with 82 and 85% activity recovery, respectively. The purified enzyme from both sources gave a single band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis.     

硫酸铯/聚乙二醇双水相体系在298.15K的相平衡

水溶性高聚物的双水相体系可用于纯化一些无机盐,获得高纯晶体。由于高聚物的存在会减小盐在溶液中的溶解度,从而可能析出晶体。本文采用自制的相平衡装置,测定了硫酸铯／聚乙二醇(PEG1000,4000,10000)双水相体系在298．15K时的等温平衡溶解度。     

One-step purification of histone deacetylase from Escherichia coli cell-lysate by counter-current chromatography using aqueous two-phase system

Aqueous-aqueous two-phase (AATP) systems composed of polyethylene glycol (PEG) (molecular mass, M(r):1000-8000) and dextran (M(r):40,000) were evaluated for purification of maltose binding protein tagged-histone deacetylase (MBP-HDAC) by counter-current chromatography (CCC). CCC purification of an MBP-HDAC from Escherichia coli cell-lysate was successfully demonstrated with a 7.0% PEG 3350-10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0. After CCC purification, both polymers in the CCC fractions were easily removed by ultrafiltration in a short period of time. The collected fractions containing target protein were analyzed by an HPLC-based in vitro assay as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis. MBP tag was digested from fusion HDAC during the CCC separation and native HDAC was purified by one-step operation with well preserved deacetyl enzyme activity.     

用吐温80—盐—水液—固萃取体系从兔肌中分离纯化乳酸脱氢酶

用吐温８０－盐－水液－固萃取体系从兔肌中分离纯化乳酸脱氢酶＊柳畅先邓江天雷锦桂游文淑李步海（中南民族学院化学系武汉４３００７４）关键词液－固萃取吐温８０乳酸脱氢酶中图分类号Ｏ６５８．２非有机溶剂液－固萃取体系是一种新的萃取体系，其特点之一是聚合物固相...     

Application of central composite design to the optimisation of aqueous two-phase extraction of human antibodies

The partition of human antibodies in aqueous two-phase systems (ATPSs) of polyethylene glycol (PEG) and phosphate was systematically studied using first pure proteins systems and then an artificial mixture of proteins containing 1mg/ml human immunoglobulin G (IgG), 10mg/ml serum albumin and 2mg/ml myoglobin. Preliminary results obtained using pure proteins systems indicated that the PEG molecular weight and concentration, the pH value and the salts concentration had a pronounced effect on the partitioning behaviour of all proteins. For high ionic strengths and pH values higher than the isoelectric point (pI) of the contaminant proteins, IgG could be selectively recovered on the top phase. According to these results, a face centred composite design was performed in order to optimise the purification of IgG from the mixture of proteins. The optimal conditions for the isolation of IgG were observed for high concentrations of NaCl and low concentrations of both phase forming components. The best purification was achieved using an ATPS containing 8% (w/w) PEG 3350, 10% (w/w) phosphate pH 6 and 15% (w/w) NaCl. A recovery yield of 101+/-7%, a purity of 99+/-0% and a yield of native IgG of 97+/-4% were obtained. Back extraction studies of IgG to a new phosphate phase were performed and higher yields were obtained using 10% phosphate buffer at pH 6. The total extraction yield was 76% and the purity 100%.