高速逆流双水相色谱法纯化卵白蛋白

生物大分子的液_固色谱纯化过程中固相载体会产生产物吸附、变性等不良影响。高速逆流色谱无需固相载体 ,且具有高分便率和高回收率的优点 ,其中有机相 水相体系在分离天然产物中应用广泛 ,而应用双水相体系分离生物大分子尚处于研究阶段。双水相高速逆流色谱体系的建立与仪器设备及操作工艺条件密切相关 ,因此利用多分离柱高速逆流色谱仪 ,研究了PEG1000-无机盐双水相体系对标准蛋白质混合物以及卵白蛋白的分离。pH值和PEG浓度对不同种类蛋白质的分配系数影响不同 ,实验发现在pH9.2的150% (W/W)PEG1000 170% (W/W)磷酸钾盐体系中 ,细胞色素C、溶菌酶和肌红蛋白的分配系数差异较大 ,且分布合理 ,因而采用该体系在 0 8mL min流速 ,85 0r min转速的条件下 ,成功分离了细胞色素C、溶菌酶和肌红蛋白的混合物。实验也发现在pH9 2的 16 0 % (W/W)PEG10 0 0 17 0 % (W/W)磷酸钾盐体系中 ,鸡蛋清样品中的主要蛋白质成分:卵转铁蛋白、卵白蛋白和溶菌酶的分配系数差异最大 ,因而采用该体系在 1 8mL min流速、85 0r mi转速的条件下,200min内从鸡蛋清样品中成功分离卵白蛋白,其电泳纯度为100%,收率为95%.     

双水相体系萃取人参根中人参皂苷的研究

建立稳定的聚乙二醇(PEG)与(NH4)2SO4双水相体系以分离人参根中人参皂苷。通过上下相体积比(R)、分配系数(K)和回收率(Y)分析双水相体系对人参皂苷的萃取效果,研究了PEG分子量、PEG/(NH4)2SO4质量分数、pH值和温度等因素对双水相成相及人参皂苷萃取的影响。结果表明:PEG分子量为3350、PEG3350的质量分数为12%、(NH4)2SO4质量分数为16%、溶液pH为7.0、温度为60℃时,双水相体系对人参皂苷有较高的萃取率,回收率可到达88.94%。     

pH调控双水相萃取色素蛋白复合体及其分配行为研究

为发展色素蛋白复合体分离纯化新方法,探究pH调控PEG1000/柠檬酸钾双水相系统萃取分离纯化色素蛋白复合体。优化萃取条件,光谱法研究其分配行为,检测产物纯度和生物活性。结果表明,最佳萃取条件为调节pH9.0,相组成CPEG100019.0%/C柠檬酸钾20.0%,蛋白质加量3.42 mg/g,K和萃取率达到最大,分别为8.8及86.0%。响应面分析法揭示,PEG1000和柠檬酸钾质量浓度及pH对分配系数和萃取率影响显著。调节pH7.0,反萃取分配系数和反萃取率最小为0.15及86.6%。蛋白质总回收率为74.2%。pH对色素蛋白复合体分配行为具有调控作用,pH大于8.5体系,色素蛋白趋于分配上相,反之分配于下相,PEG1000/柠檬酸钾以及蛋白质加入量不影响色素蛋白复合体分配于上相。电泳表征发现,萃取(pH9.0)上相存在2个蛋白质组分,相对分子量(MW)为7.0 kD及14.0 kD。反萃取(pH7.0)使相对分子量7.0 kD蛋白质组分分配于下相,该组分为LH2β亚基,经萃取和反萃取产物生物活性稳定。pH可调控PEG1000/柠檬酸钾双水相系统萃取分离色素蛋白复合体,产物纯度高,生物活性稳定。     

Geotrichum sp．SYBC WU-3脂肪酶的双水相萃取和酶学性质

初步研究双水相体系对Geotrichum sp．SYBC WU-3脂肪酶的萃取分离效果,选用PEC4000／NaH2 PO4作为戍相系统进行系统研究,考察影响脂肪酶萃取的各种因素（如PEG相对分子质量及质量分数、NaH2PO4质量浓度、pH）,并采用正交实验进一步优化实验条件,确定双水相萃取体系为PEG质量分数为30％、NaH2PO4质量分数为20％、体系pH为6,在此条件下Geotrichum sp．SYBC WU-3脂肪酶经硫酸铵沉淀和双水相萃取两步纯化的纯化倍数达到最大,较Geotrichum sp．SYBC WU-3脂肪酶粗酶纯化了22倍。Geotrichum sp．SYBC WU-3脂肪酶纯酶为低温碱性脂肪酶,最适反应温度为15oC,最适pH为9．5,相对分子质量为3．58×10^4。     

乙醇-磷酸氢二钾双水相体系萃取L-精氨酸

采用乙醇-磷酸氢二钾(K2HPO4)双水相体系萃取L-精氨酸。实验考察了乙醇浓度、K2HPO4浓度、pH、萃取温度对萃取分离L-精氨酸的影响。结果表明,L-精氨酸在该双水相体系的分配系数K随体系乙醇浓度、K2HPO4浓度的增大、萃取温度的升高而增大,随着体系pH的增大而减小;L-精氨酸在该双水相体系的萃取率随体系乙醇浓度和pH的增大而减小,随着体系K2HPO4浓度增大、萃取温度的升高而增大。     

桔梗花粉萌发与花粉管生长研究

以2年生桔梗植株为材料,采用液体培养法研究了培养基种类、PEG、蔗糖、pH以及培养温度、培养时间对桔梗花粉离体萌发生长的影响,结果表明:(1)浓度为100~150 g.L-1的PEG可显著促进桔梗花粉萌发和花粉管的生长;200~250 g.L-1PEG显著促进花粉萌发,但对花粉管生长的作用不显著。(2)100 g.L-1的蔗糖有利于花粉萌发和花粉管生长,高浓度蔗糖(200 g.L-1)有明显抑制作用;(3)桔梗花粉离体萌发和花粉管生长的适宜培养基为ME3+BK+10%蔗糖+150 g.L-1PEG(pH5.8);(4)25~40℃条件下桔梗花粉均可较好萌发,以30℃培养1.5 h为最佳培养条件。     

补增UV-B辐射对香蕉叶片光合作用和叶氮在光合碳循环组分中分配的影响

补增UV－B辐射的香蕉叶片光下呼吸速率（Rd））和不包括光下呼吸的CO2补偿点（г*）,分别为0.33μmol·m-2·s-1和46.5μl·L-1,较对照植株分别高5.6％和10.0％。在较高CO2浓度（>340μl·L-1）条件下的An/θp关系最初直线部分斜率,即表观量子产率(α-A)为0.023±0.007,而补增UV-B辐射处理的植株则降低13.0％,光能转换效率（δ）亦降低28.6％,表明UV-B辐射明显降低αA和δ。在高θp（1100μmol·m-2·s-1）和Ci<200μl·L-1条件下,对照植株的An/Ci关系为An=0.028Ci+1.44,补增UV-B辐射处理的植株则为An=0.021Ci+1.01,UV-B辐射降低羧化限制速率。最大羧化速率（Vcmax）和电子传导速率的光饱和值（Jmax）亦较低,补增UV-B辐射的叶片,叶氮在Rubisco的分配系数（PR）和叶氮在生物力能学组分的分配系数（PB）分别较对照低8.1％和3.0％,叶氮分配到类囊体膜捕光色素蛋白组分的则略见增高,UV-B辐射降低叶氮在光合循环组分的分配。     

Separation of Flavonoids from Bamboo Leaves by Aqueous Two-Phase Extraction

以竹叶黄酮水提溶液为原料,采用PEG(聚乙二醇)/(NH4 )2SO4双水相体系对竹叶黄酮进行萃取,考察了PEG平均相对分子质量、PEG质量分数、(NH4)2SO4质量分数、pH值、NaCl质量分数、原液质量分数、萃取温度等对双水相及竹叶黄酮萃取效果的影响.双水相萃取法提取竹叶黄酮的最优条件为:PEG 400 31％,(NH4)2 SO4 11％,pH3.9,NaCl 0.7％,原液51.5％,萃取温度20℃,在此条件下得到的竹叶黄酮萃取率为97.8％.结果说明,双水相萃取法操作简单方便,成本低,不会引起生物质失活或变性,适合于黄酮类化合物的萃取分离.     

两相法分离地被菊叶片质膜的研究

以地被菊(Dendranthema×grandiflorum Kitamura)叶片为材料,通过水溶性聚合物Dextran T-500和PEG 3350所构成的两相分配体系制备质膜。在一定盐浓度（5 mmol.L-1 NaCl）下选用5种不同的聚合物浓度（5.8%、6.0%、6.2%、6.4%、6.6%,W/W）,研究了地被菊叶片质膜在两相体系中的分配情况,在此基础上进一步研究了不同盐浓度（2、4、5、10、20 mmol.L-1 NaCl）对地被菊叶片质膜的纯度及蛋白产率的影响。标志酶鉴定及磷钨酸染色电镜检测的结果表明,地被菊叶片选用6.2%（W/W）聚合物浓度和7 mmol.L-1 NaCl组成的两相分配体系可获得较高纯度的密实正向型质膜囊泡。     

两相法分离地被菊叶片质膜的研究

以地被菊(Dendranthema×grandiflorum Kitamura)叶片为材料,通过水溶性聚合物Dextran T-500和PEG 3350所构成的两相分配体系制备质膜.在一定盐浓度(5mm01·L-1NaCl)下选用5种不同的聚合物浓度(5.8%、6.0%、6.2%、6.4%、6.6%,W/W),研究了地被菊叶片质膜在两相体系中的分配情况,在此基础上进一步研究了不同盐浓度(2、4、5、10、20mm01·L-1NaCl)对地被菊叶片质膜的纯度及蛋白产率的影响.标志酶鉴定及磷钨酸染色电镜检测的结果表明,地被菊叶片选用6.2%(W/W)聚合物浓度和7mm01·L-1NaCl组成的两相分配体系可获得较高纯度的密实正向型质膜囊泡.     

Optimization of bovine serum albumin partition coefficient in aqueous two-phase systems

Partitioning of proteins in aqueous two-phase systems has been shown to provide a powerful method for separating and purifying mixtures of biomolecules by extraction. These systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. There are many factors which influence the partition coefficient K, the ratio of biomolecule concentration in the top phase to that in the bottom phase, in aqueous two-phase systems. The value of the partition coefficient relies on the physico-chemical properties of the target biomolecule and other molecules and their interactions with those of the chosen system. In this work, the partition behavior of pure bovine serum albumin in aqueous two-phase systems was investigated in order to see the effects of changes in phase properties on the partition coefficient K. The concentration of NaCl and pH were considered to be the factors having influence on K. Optimal conditions of these factors were obtained using the Box-Wilson experimental design. The optimum value of K was found as 0.0126 when NaCl concentration and pH were 0.14 M and 9.8, respectively, for a phase system composed of 8% (w/w) polyethylene glycol 3,350 - 9 (% w/w) dextran 37,500 - 0.05 M phosphate at 20 °C.     

Optimization of bovine serum albumin sorption and recovery by hydrogels

Aqueous two-phase systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. Partitioning of proteins in such systems provides a powerful method for separating and purifying mixtures of biomolecules by extraction. If one of the phase forming polymers is a crosslinked gel, then the solution-controlled gel sorption may be considered as a modification of aqueous two-phase extraction. Since PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex) are common chromatographic media, we choose a PEG/dextran gel system as a model system in this study. The partitioning behavior of pure bovine serum albumin (BSA) in PEG/dextran gel systems is investigated to see the effects of variations in PEG and NaCl concentrations on the partition coefficient K. By making use of the Box-Wilson experimental design, K is shown to be maximized at 9.8 (%, w/w) PEG and 0.2 M NaCl concentrations, respectively, as 182.     

Bovine serum albumin partitioning in an aqueous two-phase system: Effect of pH and sodium chloride concentration

The partitioning of bovine serum albumin (BSA) in a polyethylene glycol 3350 (8% w/w)–dextran 37 500 (6% w/w)–0.05 M phosphate aqueous two-phase was investigated at different pHs, at varying concentrations of sodium chloride at 20°C. The effect of NaCl concentration on the partition coefficient of BSA was studied for the PEG–dx systems with initial pH values of 4.2, 5.0, 7.0, 9.0, and 9.8. The NaCl concentrations in the phase systems with constant pH value were 0.06, 0.1, 0.2, 0.3, and 0.34 M. It was observed that the BSA partition coefficient decreased at concentrations smaller than 0.2 M NaCl and increased at concentrations greater than 0.2 M NaCl for all systems with initial pHs of 4.2, 5.0, 7.0, 9.0, and 9.8. It was also seen that the partition coefficient of BSA decreased as the pH of the aqueous two-phase systems increased at any NaCl salt concentration studied.     

Production of cyclodextrin homologues using aqueous two-phase system

Cyclodextrin homologues (CDs), produced by cyclodextrin glycosyltransferase (CGTase), were simultaneously partitioned in aqueous two-phase system (ATPS). Partition coefficients of CDs were measured in PEG/salt and PEG/dextran systems. Phosphate, citrate, sulfate were tested as salt. ATPS of PEG/salt and PEG/dextran had the partition coefficients of the CDs, larger than unity. However, PEG/dextran system was observed better than PEG/salt as CGTase activity decreased sharply with salt concentration. Enzymatic reaction occurred mainly in PEG-rich bottom phase because of the low partition coefficient of CGTase. The resulting CDs transferred to the PEG-rich top phase, obeying the diffusional partition. In the ATPS of 7% PEG (M.W. 20,000) and 9% dextran (M.W. 40,000), 7 mg/ml of CDs were obtained in top phase at 4.5 hours.     

Partition of proteins in aqueous two-phase systems containing charged dextran or hydrophobic PEG

Summary The electrochemical effect of a charged dextran derivative and the hydrophobic effect of hydrophobic chain PEG derivative on partitioning of six types of proteins in PEG/dextran aqueous two-phase systems were investigated- When 1. 6%(w/w)DEAE-dextran was present in the system,the partition coefficient decreased quickly with increasing pH value;when 0. 4% (w/w)PEG pentadecanoic acid ester was present in the system, the partition coefficient of protein with strong hydrophobicity was greatly increased. The experimental results show that the influence of hydrocarbon chain PEG derivative on partition coefficient is closely related to the hydrophobicity of proteins.     

Correlation for the partition behavior of proteins in aqueous two-phase systems: effect of surface hydrophobicity and charge

Correlations to describe the effect of surface hydrophobicity and charge of proteins with their partition coefficient in aqueous two-phase systems were investigated. Polyethylene glycol (PEG) 4000/phosphate, sulfate, citrate, and dextran systems in the presence of low (0.6% w/w) and high (8.8% w/w) levels of NaCl were selected for a systematic study of 12 proteins. The surface hydrophobicity of the proteins was measured by ammonium sulfate precipitation as the inverse of their solubility. The hydrophobicity values measured correlated well with the partition coefficients, K, obtained in the PEG/salt systems at high concentration of NaCl (r = 0.92-0.93). In PEG/citrate systems the partition coefficient correlated well with protein hydrophobicity at low and high concentrations of NaCl (r = 0.81 and 0.93, respectively). The PEG/citrate system also had a higher hydrophobic resolution than other systems to exploit differences in the protein's hydrophobicity. The surface charge and charge density of the proteins was determined over a range of pH (3-9) by electrophoretic titration curves; PEG/salt systems did not discriminate well between proteins of different charge or charge density. In the absence of NaCl, K decreased slightly with increased positive charge. At high NaCl concentration, K increased as a function of positive charge. This suggested that the PEG-rich top phase became more negative as the concentration of NaCl in the systems increased and, therefore, attracted the positively charged proteins. The effect of charge was more important in PEG/dextran systems at low concentrations of NaCl. In the PEG/dextran systems at lower concentration of NaCl, molecular weight appeared to be the prime determinant of partition, whereas no clear effect of molecular weight could be found in PEG/salt systems.     

Protein partitioning in aqueous two-phase systems composed of a pH-responsive copolymer and poly(ethylene glycol)

The effect of pH and salt concentration on the partitioning behavior of bovine serum albumin (BSA) and cytochrome c in an aqueous two-phase polymer system containing a novel pH-responsive copolymer that mimics the structure of proteins and poly(ethylene glycol) (PEG) was investigated. The two-phase system has low viscosity. Depending on pH and salt concentration, the cytochrome c was found to preferentially partition into the pH-responsive copolymer-rich (bottom) phase under all conditions of pH and salt concentrations considered in the study. This was caused by the attraction between the positively charged protein and negatively charged copolymer. BSA partitioning showed a more complex behavior and partitioned either to the PEG phase or copolymer phase depending on the pH and ionic strength. Extremely high partitioning levels (partition coefficient of 0.004) and very high separation ratios of the two proteins (up to 48) were recorded in the new systems. This was attributed to strong electrostatic interactions between the proteins and the charged copolymer.     

Aqueous two-phase systems containing urea: influence on phase separation and stabilization of protein conformation by phase components.

During recombinant Escherichia coli fermentation with high expression levels, inclusion bodies are often formed. Aqueous two-phase systems have been used in the presence of urea for the initial recovery steps. To investigate phase behavior of such systems we determined phase diagrams of poly(ethylene glycol) (PEG)/sodium sulfate/urea/water and PEG/dextran T-500 (DEX)/urea/phosphate buffer/water at different concentrations of urea and different molecular weight of PEG. PEG/Na2SO4 aqueous two-phase systems could be obtained including up to 30% w/w urea at 25 degrees C and PEG/dextran T-500 up to 35% w/w urea. The binodial was displaced toward higher concentrations with increasing urea concentrations. The partition coefficient of urea was near unity. An unstable mutant of T4-lysozyme with an amino acid replacement in the core (V149T) was used to analyze the effect of phase components on the conformation of the enzyme. We showed that partitioning of tryptophan was not dependent on the concentration of urea in the phase system.     

Fundamental studies of biomolecule partitioning in aqueous two-phase systems

Phase diagram data at 4 degrees C was determined for the aqueous two-phase systems composed of polyethylene glycol, dextran, and water. The Flory-Huggins theory of polymer thermodynamics was used to correlate partitioning of biomolecules in these aqueous two-phase systems resulting in a simple linear relationship between the natural logarithm of the partition coefficient and the concentration of polymers in the two phases. This relationship was verified by partitioning a series of dipeptides which differ from one another by the addition of a CH(2) group on the c-terminal amino acid residue and by utilizing a set of low-molecular-weight proteins. The slope of the line could be expressed in terms of the interactions of the biomolecule with the phase forming polymers and water. The main result for the dipeptides was that knowledge of the partition coefficient in any of the PEG/dextran/water systems, regardless of polymer molecular weight, enabled prediction of the coefficient in all of the systems. The dipeptides were also used for determination of the Gibbs free energy of transfer of a CH(2) group between the phases. This quantity was correlated with polymer concentration, thus establishing a hydrophobicity profile for the PEG/ dextran/water systems. The methodology for predicting dipeptide partition coefficients was extended to proteins, where it was found that low-molecular-weight proteins gave a linear relationship with the tie line compositions of a phase diagram.     

Protein sorption and recovery by hydrogels using principles of aqueous two-phase extraction

Use of the thermodynamic principles of aqueous two-phase extraction (ATPE) to drive protein into a crosslinked gel is developed as a protein isolation and separation technique, and as a protein loading technique for drug delivery applications. A PEG/dextran gel system was chosen as a model system because PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex(R)) are common chromatographic media. The effects of polymer concentrations and molecular weights, salts, and pH on the partitioning of ovalbumin matched ATPE heuristics and data trends. Gel partition coefficients (Cgel/Csolution) increased with increasing PEG molecular weight and concentration and decreasing dextran concentration (increased gel swelling). The addition of PEG to the buffer solution yielded partition coefficients more than an order of magnitude greater than those obtained in systems with buffer alone, or added salt. A combined salt/PEG system yielded an additional order of magnitude increase. For example, when ovalbumin solution (2.3 mg/mL) was equilibrated with Sephadex(R) G-50 at pH 6.75, the partition coefficients were 0.13 in buffer, 0.11 in buffer with 0.22M KI, 2.3 in 12 wt% PEG-10,000 and 32.0 in 12 wt% PEG-10, 000 with 0.22M KI. The effect of anions and cations as well as ionic strength and pH on the partitioning of ovalbumin also matched ATPE heuristics. Using the heuristics established above, partition coefficients as high as 80 for bovine serum albumin and protein recoveries over 90% were achieved. In addition, the wide range of partition coefficients that were obtained for different proteins suggests the potential of the technique for separating proteins. Also, ovalbumin sorption capacities in dextran were as high as 450 mg/g dry polymer, and the sorption isotherms were linear over a broad protein concentration range.