Partition of tannery wastewater proteins in aqueous two-phase poly (ethylene glycol)-magnesium sulfate systems: Effects of molecular weights and pH

The partitioning behavior of soluble proteins from tannery wastewater using aqueous two-phase system (ATPS) was investigated. An ATPS polyethylene glycol (PEG)/MgSO₄ was examined with regard to the effects of PEG molecular weight (MW) and concentration, MgSO₄ concentration, pH and NaCl concentration on protein partition and extraction. The partition coefficients measured for soluble proteins were proportional to the difference in PEG concentration between the phases. The MW and concentration of PEG were found to have significant effects on protein partition and extraction with low MW PEG4000 showing the best conditions for the partitioning of protein in PEG+MgSO₄+water system. Sulfate salt was chosen as the phase-forming salt because of its ability to promote hydrophobic difference between the phases. This system was operated at room temperature . Increase in pH of the system increases the partition coefficient of proteins from tannery wastewater. The addition of sodium chloride showed significant influence on the partition coefficient. ATPS comprising PEG4000-magnesium sulfate provided a means for the recovery of proteins from tannery wastewater. The maximum percentage yield of protein extracted is 82.68%.     

Extraction of microbial transglutaminase from <Emphasis Type="Italic">Amycolatopsis</Emphasis> sp. fermentation broth using aqueous two-phase system

Partitioning of microbial transglutaminase (MTG) from Amycolatopsis sp. in the polyethylene glycol (PEG)/salt-based ATPS was investigated for the first time. The key parameters such as the molecular weight of PEG (PEG 600-6000), the type and concentration of phase-forming salt (ammonium sulfate or phosphates), the pH of system (pH 5.0-8.5), and the concentration of neutral salt (0-6% NaCl, w/w) were determined. The partition coefficient of the enzyme was not linear with PEG molecular weight; PEG1000 gave better yield than others. The concentration of PEG1000, ammonium sulfate and NaCl, and the system pH showed effects with different extents on specific activity (SA) and yield of the enzyme. In the ATPS of 26% w/w PEG 1000 and 19% w/w ammonium sulfate in the presence of 5% w/w NaCl and at pH 6.0, MTG was partitioned into the PEG-rich phase with a maximum yield of 86.51% and SA was increased to 0.83. The results of SDS-PAGE showed the MTG produced by the test strain differed from the enzymes reported before. Thus, this study proves that ATPS can be used as a preliminary step for partial purification of MTG from Amycolatopsis sp. fermentation broth.     

金属螯合双水相亲和分配技术分离纳豆激酶的研究

利用金属螯合亲和双水相分配技术对纳豆激酶的分离纯化进行了研究。考察了双水相系统、聚合物的分子量和浓度、亲和配基加入量、pH值、相比以及生物质加入量等因素对亲和分配的影响。结果表明,双聚合物系统比聚合物／无机盐系统更有利于纳豆激酶亲和分配;pH值和亲和配基加入量是影响分配的关键因素。优化的分配条件为：2．6％聚乙二醇,20．2％羟丙基淀粉,5％亲和配基PEG-IDA—Cu(Ⅱ),相比12,pH8．2,发酵液加入量15％。分配系统放大到100g,仍保持一致的酶活收率(90％)和纯化因子(2.0)。设计了两次分配分离流程,纯化因子达到3．52,总收率为81％。     

Bovine serum albumin partitioning in polyethylene glycol (PEG)/potassium citrate aqueous two-phase systems

The extraction and back-extraction of bovine serum albumin (BSA) have been studied by liquid–liquid extraction with poly(ethylene glycol) (PEG)/potassium citrate aqueous two-phase system (ATPS). In this work, the ATPS was examined with regard to the effects of PEG molecular weight (PEG 1000, 2000, 4000 and 6000), PEG and potassium citrate concentration, BSA concentration (C_BSA) and pH on BSA partition. The pH was found to have significant effects on BSA partition with low molecular weight PEG 1000. The yield of the BSA, 99%, was obtained in the top phase under the following conditions: 19% (w/w) PEG 1000, 20% (w/w) potassium citrate and 0.75 mg/g C_BSA at pH 7.0 and 30 °C. BSA can be re-extracted to a new citrate phase by decreasing the pH of the system with a 92% yield. The back-extraction not only separates the BSA from the polymer, but also allows the polymer to be recycled. The global yield (Y_e + Y_be) is up to 91%.     

Recovery and partial purification of thermophilic β-xylosidase derived from recombinant Bacillus megaterium MS941 by aqueous two-phase system

A polymer–salt-based aqueous two-phase system (ATPS) was developed for the effective extraction and purification of extracellular β-xylosidase from the fermentation broth of recombinant Bacillus megaterium MS941. The effect of molecular weight (MW) of polyethylene glycol (PEG), tie-line length (TLL), volume ratio (V_R), crude loading and pH on the recovery performance was evaluated. Under the optimal extraction conditions, β-xylosidase was successfully purified up to 23-fold with a recovery yield of 99% in the bottom salt-rich phase at PEG 4,000/potassium phosphate ATPS comprising TLL of 41.8, V_R of 2.3, crude loading (C_L) of 30% (w/w) at pH 6.     

PEG-(NH_4)_2SO_4双水相萃取法提取壳聚糖酶的研究

采用PEG-(NH4)2SO4双水相体系直接从Bacillussp.LS发酵液上清液中分离壳聚糖酶。研究了体系中PEG分子量、PEG质量分数、(NH4)2SO4质量分数、NaCl质量分数和pH值对壳聚糖酶分配系数及萃取率的影响。结果表明,室温下双水相萃取最佳条件为:PEG600 20%、(NH4)2SO420%、NaCl 0.1%、pH值6.0,在此条件下壳聚糖酶分配系数达5.91,萃取率达88.7%。     

Separation and Purification of Aloe Anthraquinones Using PEG/Salt Aqueous Two-Phase System

The anthraquinones were extracted from Curacao aloe leaves. Aqueous two-phase system (ATPS) of polyethylene glycol (PEG)/salt, coupled with spectrophotometry and high performance liquid chromatography (HPLC) were employed for the first time as an attractive alternative for the downstream processing of aloe anthraquinones, mainly for the removal of the impurities without additional steps. The influence factors such as molecular mass and concentration of PEG, type, and concentration of neutral salt, temperature, and pH on the phase partition behavior of ATPS had been studied. Under the optimal condition, the highest extraction yield 90.54% was obtained in PEG phase using PEG-6000/(NH₄)₂SO₄ system to a mass ratio of 2:1 at 40°C, pH 3.0 with 0.6 g sodium chloride added. The reverse extraction of anthraquinones from the PEG phase was achieved with a recovery of 70.15% by adjusting the pH. Meanwhile, the PEG could be recycled. The major components in aloe anthraquinones of aloe-emodin and chrysophanol were analyzed by HPLC before and after ATPS extraction process. Compared with conventional purification methods, this technique can be completed in one operation; besides it is low-cost and environmentally friendly.     

Optimization of Serine Protease Purification from Mango (Mangifera indica cv. Chokanan) Peel in Polyethylene Glycol/Dextran Aqueous Two Phase System

Mango peel is a good source of protease but remains an industrial waste. This study focuses on the optimization of polyethylene glycol (PEG)/dextran-based aqueous two-phase system (ATPS) to purify serine protease from mango peel. The activity of serine protease in different phase systems was studied and then the possible relationship between the purification variables, namely polyethylene glycol molecular weight (PEG, 4000-12,000 g·mol(-1)), tie line length (-3.42-35.27%), NaCl (-2.5-11.5%) and pH (4.5-10.5) on the enzymatic properties of purified enzyme was investigated. The most significant effect of PEG was on the efficiency of serine protease purification. Also, there was a significant increase in the partition coefficient with the addition of 4.5% of NaCl to the system. This could be due to the high hydrophobicity of serine protease compared to protein contaminates. The optimum conditions to achieve high partition coefficient (84.2) purification factor (14.37) and yield (97.3%) of serine protease were obtained in the presence of 8000 g·mol(-1) of PEG, 17.2% of tie line length and 4.5% of NaCl at pH 7.5. The enzymatic properties of purified serine protease using PEG/dextran ATPS showed that the enzyme could be purified at a high purification factor and yield with easy scale-up and fast processing.     

Application of Response Surface Methodology for Optimization of Bromelain Extraction in Aqueous Two-Phase System

Extraction of bromelain from pineapple fruit in an aqueous two phase system (ATPS) composed of polyethylene glycol (PEG) 1500 and potassium phosphate has been studied using response surface methodology. The various process variables such as PEG, potassium phosphate and NaCl concentration, and pH were optimized using a central composite rotatable design (CCRD) of response surface methodology (RSM) based on the partition coefficient, % yield, and purification factor of an enzyme. An optimized ATPS composed of 14% (w/w) PEG 1500, 17.66% (w/w) potassium phosphate and 1 mM sodium chloride at pH 7.5 was used to purify bromelain from a pineapple fruit. With this system, a maximum enzyme partition coefficient of 12.62 and %yield of 90.33 in the top PEG-rich phase with a purification factor of 2.4 was predicted. The enzyme partition coefficient, % yield, and purification factor obtained from experimentation are 12.22, 89.65, and 2.8, respectively, in the top PEG phase. The response model is validated by the closeness between the predicted and experimental results.     

Response surface methodology for the evaluation of guanidine hydrochloride partitioning in polymer-salt aqueous two-phase system

The current study employed response surface methodology (RSM) with a face-centered central composite design (CCD) to indicate the essential variables on the partition coefficient of guanidine hydrochloride (GuHCl) in the poly (ethylene glycol) (PEG)-phosphate aqueous two-phase system (ATPS). To evaluate the partition coefficients of GuHCl in the mentioned ATPS, the pH (7.0, 8.5 and 10.0), GuHCl concentration (1.0, 3.5 and 6.0% w/w), PEG molecular weight (2,000, 4,000 and 6,000 gmol^?1) and PEG/potassium phosphate concentrations ratio were selected as independent variables. A quadratic model is suggested to find the impact of these variables. The suggested model has a strong harmony with the experimental data. The results of the model display that the GuHCl concentration and weight percent of the salt in feed have a large and small influence on the GuHCl partitioning.     

Recovery of Human Interferon Alpha-2b from Recombinant Escherichia coli by Aqueous Two-Phase System

Recovery of periplasmic human recombinant interferon alpha-2b (IFN-α2b) from Escherichia coli rosetta-gami2 (DE3) using a single-step polyethylene glycol (PEG)-potassium phosphate aqueous two-phase system (ATPS) was investigated in this study. The influences of system parameters including PEG molecular weight, tie-line length, volume ratio, crude stock loading, system pH, and sodium chloride (NaCl) concentration (%, w/w) were studied. The results showed that the optimum condition to obtain the high purification factor of IFN-α2b in a single step was achieved by ATPS composed of 4% (w/w) PEG 8000, 13% (w/w) potassium phosphate, 0.5% (w/w) NaCl, 10% (w/w) crude stock, and a system pH of 6.5. A purification factor of 26.3 and recovery yield of 40.7% were obtained from optimized ATPS.     

Liquid-liquid extraction of protease from cold-adapted yeast Rhodotorula mucilaginosa L7 using biocompatible and biodegradable aqueous two-phase systems

This work aimed to optimize the extraction of an extracellular protease produced by the cold-adapted yeast Rhodotorula mucilaginosa L7 using aqueous two-phase systems (ATPS) comprising polyethylene glycol (PEG) and sodium citrate or sodium tartrate. First, the biocompatibility of the phase forming agents was assessed. The results obtained with PEG-2000, PEG-4000, and PEG-6000 demonstrated that even at large PEG concentrations (32 wt%) the protease maintains its activity after 3 h of reaction, whereas an increase in salt concentration provokes a gradual decrease in protease stability. Subsequently, the partitioning of the protease in both types of ATPS was assessed, evaluating the effect of temperature, molecular weight, and concentration of PEG on protease purification, using two 2³-full factorial designs. The best partitioning conditions were obtained in PEG-6000/sodium tartrate-based ATPS, at 30ºC (with a yield of 81.09 ± 0.66% and a purification factor of 2.51 ± 0.03). Thus, considering the biodegradable characteristics of the system, the PEG/sodium tartrate ATPS is a viable and economic low-resolution step in protease purification, with a strong potential for future industrial application.     

双水相分配结合温度诱导相分离从酵母中提取谷胱甘肽

对用双水相分配技术结合温度诱导相分离技术从酵母中提取谷胱甘肽（GSH）进行了研究．考察了GSH在环氧乙烷──环氧丙烷无规共聚物（EOPO）／羟丙基淀粉（PES）系统中的分配行为,包括两次分离过程（双水相分配及温度诱导过程)中的不同系统组成、pH等对GSH分配的影响,确定了较佳的双水相系统──EOPO400013％,PES10010％,pH=10.5．在此基础上进行了从酵母细胞中提取GSH的工艺研究,设计了合理的分离流程,研究结果表明GSH的总萃取率可达80％以上．     

Aqueous two‐phase systems for the recovery of a recombinant viral coat protein from Escherichia coli

In this study the use of an aqueous two‐phase system (ATPS) following the direct chemical extraction of a recombinant viral coat protein, from the cytoplasm of Escherichia coli, is evaluated. The driving force is the need to establish an economically‐viable process for the manufacture of a vaccine against human papilloma infection. The partition behaviour of recombinant L1 protein, the major structural protein of the virus, and DNA was investigated in a polyethylene glycol (PEG)–phosphate system. An evaluation of system parameters including PEG molecular mass and the concentrations of PEG and phosphate was conducted, to estimate conditions under which the L1 protein and DNA partition to opposite phases. ATPS extraction comprising a volume ratio of 1.00, PEG 1000 (18.0%(w/w)) and phosphate (15.0%(w/w)) provided the conditions for accumulation of DNA into the bottom phase and concentration of L1 protein into the opposite phase (ie partition coefficient of DNA; ln K_DNA < 0.0 and partition coefficient of L1; ln K_L1 > 2.5). The findings reported here demonstrate the potential of ATPS to recover recombinant protein released from E coli by direct chemical extraction. © 2002 Society of Chemical Industry     

蒲公英总黄酮在聚乙二醇-硫酸铵双水相体系中的分配与提取

蒲公英中内含的黄酮类物质具有较高药用价值,用双水相萃取法提取植物中有效成分是新型提取高附加值生物质的有效方法。本文考察了PEG/(NH4)2SO4双水相体系萃取分离蒲公英总黄酮时聚乙二醇相对分子质量、PEG质量分数、(NH4)2SO4质量分数、温度、pH值5个因素对分配行为的影响,并通过正交实验优化了工艺条件。结果表明最佳双水相提取工艺条件为：(NH4)2SO4质量分数18%,PEG1000质量分数23%,pH值5.34,提取温度25℃,NaCl盐的存在与否对萃取影响很小,蒲公英中总黄酮的提取率可达5.47%。因此,使用双水相法提取蒲公英总黄酮是该类提取技术中一种更加绿色环保和高效的方法。     

Partition of alkaline protease in aqueous two-phase systems of polyethylene glycol 1000 and potassium phosphate

This article presents a study of polyethylene glycol 1000 (PEG1000)/potassium phosphate aqueous two-phase systems (ATPSs) forBacillus subtilis NS99 alkaline protease extraction. The objectives were to evaluate effects of system pH (7.5, 8.5,9.5, and 10.5), and NaCl concentration (0,4,7, and 10% (w/w)) on ATPS binodal curves, effects of system pH, NaCl concentration, and tie-line length (TLL) on alkaline protease partition coefficient (K) and yield (Y%) at room temperature (30±2 ‡C). Casein hydrolysis was used for determination of alkaline protease activity. It was revealed that system pH had the slightest effect on locations of binodal curves (except at pH 10.5). In contrast, addition of NaCl appeared to have a significant effect on phase characteristics since binodal curves of systems with NaCl (4-10% (w/w)) shifted significantly towards the origin in comparison to the ones without NaCl. Increased NaCl concentration from 4 to 10% (w/w), however, showed trivial influence on locations of the binodal curves. Changes of system compositions due to variation in system pH, TLL, and NaCl concentrations obviously resulted in varied obtainable K and Y% of alkaline proteases. Longer TLL and higher pH generally resulted in higher K. In contrast, the lower NaCl concentration, the higher K. Since the same phase volume ration (1:1) was used throughout the experiments, Y% depended solely on K. The most suitable PEG1000/potassium phosphate ATPS was determined at pH 9.5, and comprised PEG1000, potassium phosphate, and NaCl 18.0,13.0, and 0% (w/w), respectively. This system resulted in considerably high K, and Y% of 20.0, and 95.1%, respectively. Information from this study will be important for further development of an ATPS extraction unit for alkaline protease recovery.     

Partitioning of Propionic Acid in Polyethylene Glycol/ Different Salts of Sulfate Aqueous Two-Phase Systems

The extraction of propionic acid has been studied by liquid–liquid extraction with poly ethylene glycol /different salts of sulfate aqueous two-phase system (ATPS). In this study, a multilevel factorial design was applied to evaluate the effects of four independent variables (PEG molecular weight, type of salt, salt concentration, and temperature) on propionic acid extraction yield. The statistical significance of each factor was evaluated by an analysis of variance. The results indicated that the type and concentration of salt were the major contributing factors to propionic acid extraction. The PEG4000/ MnSO₄ system was suitable for the extraction of propionic acid from dilute water.     

PEG/（NH_4）_2SO_4双水相萃取分离茶氨酸的研究

采用聚乙二醇（PEG）/（NH4）2SO4双水相体系萃取分离生产茶多酚所得废液中的茶氨酸,考察了PEG分子量与含量、硫酸铵含量、pH、温度、加盐（KCl、KBr、KI）、茶氨酸含量对双水相及萃取分离茶氨酸的影响。结果表明,PEG/（NH4）2SO4双水相萃取分离茶氨酸的适宜条件是：PEG平均分子量为4 000,质量分数为10%,硫酸铵质量分数为15%,pH约为6,30℃。在此条件下,茶氨酸的分配系数K1=0.16,蛋白质的分配系数K2=0.28,糖类的分配系数K3=9.8,茶氨酸在下相的萃取率为89.5%,可以将茶多酚废液中的茶氨酸与糖类及其他有颜色的杂质分开。     

Recovery of value‐added globular proteins from tannery wastewaters using PEG – salt aqueous two‐phase systems

Leather processing involves discharge of high‐value soluble globular proteins in the wastewater. The recovery of value‐added products from the wastewaters is gaining more importance in the context of recovery of wealth from waste. The recovery of these globular proteins from tannery wastewater was selected as a practical model system to study the implementation of polyethylene glycol (PEG)‐sulfate aqueous two‐phase systems (ATPS). The partition coefficient of bovine serum albumin is comparable to that of soluble proteins from tannery wastewaters. The influence of concentration of polymer, salt, pH and temperature on the partitioning of soluble proteins from tannery wastewaters has been studied. The PEG6000 + sodium sulfate + water system provide better partitioning of these soluble proteins as compared to PEG6000 + ammonium sulfate system. The maximum protein recovery yield for PEG6000 + sodium sulfate + water system at 20 °C is 92.75%. The influence of temperature indicates the recovery of proteins from tannery wastewater to be better at lower temperature. The findings of these studies raise the potential application of ATPS processes for protein recovery from complex biological systems. Copyright © 2006 Society of Chemical Industry     

Aqueous two-phase extraction of punicalagin (α+β) from pomegranate peel by response surface methodology

In order to enhance the recovery of punicalagin from pomegranate peel, extraction of punicalagin (α + β) was studied using aqueous two-phase system. Box Behnken design of response surface methodology was used as a tool to study the effect of Poly Ethylene Glycol (PEG) concentration, PEG molecular weight, salt concentration, solute amount and pH on the extraction yield of punicalagin. The optimal conditions were found to be 18% (w/v) PEG concentration, 8000 PEG molecular weight, 16% (w/v) salt concentration, 0.5 g solute amount and 6 pH. Under these conditions, experimental yield and predicted yield was found to be 43% and 48%, respectively.