Affinity Partitioning Of Enzymes Using Unbound Triazine Dyes In Peg/Phosphate System

Abstract

The partitioning of alcohol dehydrogenase glucose-6-phosphate dehydrogenase and hexokinase from pressed bakers' yeast was studied in polyethylene glycol (PEG)/potassium phosphate aqueous two-phase system using unbound reactive triazine dyes as affinity ligands. The various parameters investigated were ligand type and concentration, pH of the system, phase composition of the system, and molecular weight of PEG. It was found that the best ligands gave an increase in partition coefficient of 2 to 3 times over that in the absence of the ligand.     

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％。     

Purification of Glucose Oxidase and β‐Galactosidase by Partitioning in a PEG‐Salt Aqueous Two‐Phase System in the Presence of PEG‐Derivatives

Abstract

Purification of glucose oxidase from Aspergillus niger and that of β‐galactosidase from Kluyveromyces lactis have been attempted using poly(ethylene glycol) (PEG)‐sodium sulfate aqueous two phase system (ATPS) in the presence of PEG‐derivatives, i.e. PEG‐Coomassie brilliant blue G‐250 and PEG‐benzoate, PEG‐palmitate and PEG‐TMA, respectively. The enzymes showed poor partitioning towards the PEG phase in comparison with other proteins in ATPS containing no ligands. Selective partitioning of other proteins was observed towards the PEG phase in the presence of PEG‐benzoate and PEG‐palmitate enriching β‐galactosidase in the salt phase whereas in the case of glucose oxidase, PEG‐Coomassie brilliant blue G‐250 derivative worked as a better affinity ligand for other proteins. A 19‐fold purification was obtained with the PEG dye derivative after 5 stage cross extractions with 80% recovery of glucose oxidase and an enrichment factor upto ～7 for β‐galactosidase with the PEG‐TMA derivative. The interaction of PEG‐benzoate and PEG‐TMA ligands with the active site of β‐galactosidase has been evaluated by molecular modeling. The effect of the molecular weight of glucose oxidase on its partitioning was confirmed as the molecular simulation shows strong affinity interaction of PEG‐glucoside with the enzyme.     

Aqueous Two‐Phase Partitioning of Glucose Isomerase from Actinoplanes missouriensis in the Presence of PEG‐Derivatives and its Immobilization on Chitosan Beads

Abstract

Purification of glucose isomerase by its partitioning in a PEG‐salt aqueous two‐phase system (ATPS) in the presence of PEG derivatives has been studied. Selective partitioning of the proteins was observed towards the PEG phase containing PEG‐benzoate and PEG‐palmitate, enriching glucose isomerase in the salt phase. Cross‐current extraction in 4 stages in the presence of PEG‐palmitate gave an enrichment factor of ～5 for the enzyme. After initial purification with ATPS, glucose isomerase was immobilized on cross‐linked chitosan beads. The immobilized enzyme was stable over a wider pH range (5.2–9.0) and showed an optimum pH of 6.5     

Recovery of Recombinant Dog Gastric Lipase from Corn Endosperm Extract

Abstract

Two separation methods, aqueous two‐phase (ATP) partitioning and cation‐exchange chromatography, were compared as alternative methods for the recovery of recombinant dog gastric lipase (r‐DGL) from extracts of transgenic corn endosperm. r‐DGL is a hydrophobic, acid‐stable protein targeted for stable expression in endosperm. Polyethylene glycol (PEG) ‐ salt ATP system parameters of PEG molecular weight, phase‐forming salt, NaCl addition, Triton X‐100 concentration and phase ratio were adjusted to achieve favorable partitioning. The purification factor and yield of r‐DGL in the bottom phase of a PEG 3350 (14.2%)‐Na₂SO₄ (8.5%)‐NaCl (0.5%)‐Triton X‐100 (2 mM) system at pH 4 were 1.5 and 80%, respectively. A higher purification factor of 2.3 and nearly 100% yield of r‐DGL was obtained in the top phase of a PEG 3350 (9.4%)‐phosphate (14.3%)‐NaCl(1.5%)‐Triton X‐100 (2 mM) system at pH 4.0. The yield, purification factor, and concentration factor were 90%, 7.7, and 3.6, respectively, for the alternative of cation‐exchange on CM‐Sepharose. Countercurrent ATP partitioning with 3–7 stages was calculated to achieve a purification factor equivalent to that from cation exchange but with a lower concentration factor. While the cation exchange was favored on this basis, the two approaches were close enough that further optimization and economic analysis would be needed to be definitive.     

Optimization of recovery of esterase from Serratia marcescens using combination of the solvent impregnated resin and aqueous two-phase extraction techniques

ABSTRACT

The performance of tunable aqueous polymer phase impregnated resins (TAPPIR) which is the combination of the solvent impregnated resin principle and an aqueous two-phase system for the separation of esterase from Serratia marcescens was evaluated in this study. Different molecular weight of polyethylene glycol (PEG) (2000, 4000 and 6000) at concentration ranging from 5% to 20% (w/w) and potassium citrate were used to construct the aqueous phase in TAPPIR technology. Optimum composition of PEG and salt for esterase partitioning was determined using response surface methodology. The optimum condition for the purification of esterase was impregnation of 25% (w/w) of PEG 2000 into 4 mm porous glass beads and extraction of esterase using 15% (w/w) potassium citrate at pH 8 containing 12% (w/w) crude loading with the addition of 4% (w/w) NaCl. Esterase from S. marcescens was successfully purified by the TAPPIR technology up to 5.32 of purification factor with a yield of 75.98%.     

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.     

PEG-BASED AQUEOUS BIPHASIC SYSTEMS AS IMPROVEMENT FOR KRAFT HARDWOOD PULPING PROCESS

Aqueous biphasic systems (ABS) represent wholly aqueous systems that are safe, nontoxic, nonflammable, and relatively environmentally benign as extraction media. We have studied detailed phase diagrams and partitioning data currently used in wholly aqueous solvent extraction or reactive extraction processes that may be applicable to the delignification of cellulosic materials at elevated temperature. The study results indicate that increasing the concentration of salt improves the degree of phase divergence. The partitioning results of different lignin species in PEG/salt ABS show that lignin prefers the polymer-rich phase of ABS. Four complete time-at-temperature (130-160°C) batch-cooking experiments were made using a Kraft pulping solution with and without PEG. The results for wood chips composed of a mixture of southern hardwood indicate that the use of PEG results in a pulp with lower Kappa number, lower lignin content, higher residual pulp yield, and reduced alkali consumption.     

Affinity Partitioning and Its Potential in Biotechnology

Abstract

The affinity partitioning technique and its applications to biotechnological separations are reviewed. In spite of the great potential for large-scale continuous processing and the wide success in laboratory separations offered by the technique, relatively few reports of biotechnological applications have been made. This underutilization is attributed in part to the lack of design criteria for establishing and optimizing partitioning systems. A first step toward developing a thermodynamically based design model for affinity partitioning, based on the incorporation of a Gibbs energy model for phase equilibria in polymer solutions into a separate theory accounting for the affinity effect, is presented.     

Purification of Glutaminase from Zygosaccharomyces rouxii in Polyethylene Glycol– Sodium Sulphate Aqueous Two-Phase System

L-glutaminase (EC 3.5.1.2) produced from Zygosaccharomyces rouxii NRRL-Y 2547 was partitioned in an aqueous two phase system comprising PEG 2000 and sodium sulphate. The effects of tie line length (TLL), pH, broth loading (BL), volume ratio, and neutral salt concentration on enzyme partitioning and purification were investigated. The optimal condition for the partitioning of glutaminase was obtained through response surface methodology and obtained the partition coefficient and yield of 12.99 and 95.12%, respectively. The purification factor of 5.59 and selectivity of 6.52 were achieved at the optimal condition.     

Flowsheet simulation of aqueous two‐phase extraction systems for protein purification

BACKGROUND: Aqueous two‐phase extraction (ATPE) has many advantages as an efficient, inexpensive large‐scale liquid–liquid extraction technique for protein separation. However, the realization of ATPE as a protein separation technology at industrial scales is rather limited due to the large, multidimensional design space and the paucity of design approaches to predict phase and product behavior in an integrated fashion with overall system performance. This paper describes a framework designed to calculate suitable flowsheets for the extraction of a target protein from a complex protein feed using ATPE. The framework incorporated a routine to set up flowsheets according to target protein partitioning behavior in specific ATPE systems and a calculation of the amounts of phase‐forming components needed to extract the target protein. The thermodynamics of phase formation and partitioning were modeled using Flory‐Huggins theory and calculated using a Gibbs energy difference minimization approach. RESULTS: As a case study, suitable flowsheets to recover phosphofructokinase from a simple model feedstock using poly(ethylene glycol)‐dextran (PEG6000‐DxT500) and poly(ethylene glycol)‐salt (PEG6000‐Na₃PO₄) two‐phase systems were designed and the existence of feasible solutions was demonstrated. The flowsheets were compared in terms of product yield, product purity, phase settling rate and scaled process cost. The effect of the mass flowrates of phase‐forming components on product yield and purity was also determined. CONCLUSION: This framework is proposed as a basis for flowsheet optimization for protein purification using ATPE systems. Copyright © 2010 Society of Chemical Industry     

Experimental studies on phase equilibria of PEG-water-dextran/ammonium sulfate systems and partitioning of albumin into two water-continuous phases

The clouding points and equilibrium concentrations of PEG/dextran/water and PEG /ammonium sulfate/water systems were experimentally determined for different molecular weights of PEG by titration method and direct determination of concentrations. In phase diagrams of PEG-water with ammonium sulfate or dextran, the addition of salt or dextran induced the phase separation of PEG-rich phases near the PEG-lean phases having the different partitioning of PEG. The concentrations of PEG in PEG-rich phases increase as the amounts of PEG or salt and dextran increase, while the concentrations of PEG in aqueous media decrease in any cases. The higher the molecular weight of PEG has, the wider the two-phase regions are. In dextran DT10 systems, the partition coefficients of egg albumin into PEG-lean phases increase with concentrations of dextran and the molecular weight of PEG. In ammonium sulfate systems, the partitioning coefficients showed a maximum, having lower partitioning at the very high and low concentration of salts. It is also observed that as the amounts of albumin increase, the partitioning of albumin into PEG-lean phase increases.     

Affinity Partitioning of Metal Ions in Aqueous Polyethylene Glycol/Salt Two-Phase Systems with PEG-Modified Chelators

Abstract

The systematic partitioning of metal ions in the presence of polyethylene glycol (PEG) chelate derivatives in PEG/salt two-phase systems is described. Results show that the partitioning is enhanced significantly, and this effect is apparently based on the characteristics of binding of the chelate and the nature of the metal ion in addition to the effect of the attached PEG molecule. Previous work with PEG-iminodiacetic acid has been extended with the chelating derivatives PEG-TED [tris(carboxymethyl)ethylene diamine], PEG-TREN-Cm [carboxy-methylated-tris(2-aminoethyl)amine], and PEG-ASP (aspartic acid). Their effectiveness to selectively partition the metal ions is demonstrated and discussed. The partition experiments were performed using two-phase systems of PEG (8000 MW), sodium sulfate, the PEG-chelating derivatives, and different concentrations of copper, nickel, and cobalt as model metal ions. Partition coefficients (ratio of top to bottom metal concentrations) greater than 1 were obtained for the metal ions with all the chelating derivatives tested. Selective enhancement of partitioning was observed in all cases. Based on the experimental results and theoretical principles, the method shows potential as an alternative mode to remove efficiently and selectively metal ions from solutions using aqueous two-phase systems and PEG-chelate derivatives.     

Acid-base equilibria of sulfonephthalein dyes in aqueous polymer-surfactant media

The acid-base equilibria of three anionic sulfonephthalein dyes, viz., bromothymol blue (BTB), thymol blue (TB), and cresol red (CR), were studied spectroscopically in aqueous media containing the water-soluble noninonic polymers polyvinyl alcohol (PVA) and polyethylene glycol (PEG) in the presence of an anionic surfactant, sodium dodecyl sulfate (SDS). A partition equilibrium method was used to determine the equilibrium constant of partition of the dyes between the micellar pseudo phase and aqueous phase in the presence of PVA and PEG. The critical aggregation concentrations (CAC) of the surfactants in buffered aqueous systems containing the neutral polymers were also determined. The CAC of the polymer-surfactant systems were found to be lower than the critical micelle concentration of such systems in the absence of polymer, in otherwise identical conditions. The pH-dependent association constants, K _ass, of the sulfonephthalein dyes with the SDS-PVA system increased with the increase in molecular weight of the polymer. The interactions of the dyes with the buffered aqueous SDS-PVA and SDS-PEG systems were found to be endothermic and entropy oriented. In the polymer domain, the head group region of the micelles was more exposed at lower concentrations of the polymer, but under excess polymer concentration they were increasingly shielded, which impaired their electrostatic interaction with the dyes.     

PEG-400 Partitioning in the HCCD/PEG Process for Cs and Sr Recovery

Abstract

The properties of the chloro-protected cobalt bis(dicarbollide) anion in the acidic form (HCCD), and in the presence of polyethylene glycol (PEG-400), are well known for the recovery of Cs and Sr from acidic radioactive streams. In the early development of HCCD/PEG extraction processes, questions were raised regarding the ability to control the concentration of PEG-400 in the organic phase due to its high solubility in the aqueous process solutions relative to HCCD or the diluent. The purpose of this study was to quantify the partitioning behavior of PEG-400 under a wide variety of relevant process conditions. PEG distribution ratios (D _PEG ) were measured by equilibrium batch contacts between the organic and aqueous phases over a wide range of conditions using radiometric techniques with ¹⁴C labeled PEG-400 to monitor the behavior of the bulk material. The results vary dramatically from 0.1 < D_PEG < 50, indicate that the PEG phase transfer kinetics are rapid, and that the aqueous phase nitric acid concentration has minimal impact on PEG solubility. The molar concentration ratio of [HCCD]:[PEG] in the organic phase has the greatest impact on PEG solubility. This ratio should be maintained at [HCCD]:[PEG] greater than or equal to approximately 6 to minimize PEG losses from the organic phase.     

多元渗透维里方程通式的表达：双水相系统的液液平衡计算

提出了基于三元系修正Ｐｉｔｚｅｒ模型的多元渗透维里方程通式，并应用于双水相系统液液平衡性质的关联和预测，用汽液平衡数据关联模型参数，预测了聚乙二醇／硫酸镁双水相系统的液液平衡；用汽液平衡数据和尽可能少的液液平衡数据关联模型参数。同时预测了焦点或不含电解质的ＰＥＧ／葡萄糖双水相系统的液液平衡和ＫＣｌ电解质在ＰＥＧ／ＤＥＸ系统中的分配系数。计算结果表明，模型有较好的预测功能。     

The use of reactive dyes in protein separation processes

The salient points of the chemistry of triazine based reactive dyes judged to be of interest to biochemists are summarised and the various alternative reactive systems and chromophores used in reactive dyes outlined. The development of the use of reactive dyes in dye affinity chromatography is discussed, parallels being drawn between textile dyeing and immobilisation techniques to provide suggestions for improvements in technique. The properties of the matrix are considered and structural features in the dye responsible for protein attraction analysed. The recent tailoring of dye structures to enhance utility in dye affinity chromatography and progress in the scaling up of high performance liquid affinity chromatography is reviewed.     

Modeling the protein partitioning in aqueous polymer two-phase systems: influence of polymer concentration and molecular weight

Aqueous polymer two-phase system provides a powerful method for separation and purification of biomaterials. Among various factors, polymer concentration and polymer molecular weight are essential and have strong impact on the protein partitioning in these systems. Based on the modified Pitzer's model, a simple expression has been obtained for correlating protein partitioning in aqueous polymer two-phase systems with varying polymer concentration and different polymer molecular weights. Using only one group of parameters for each target protein, the partition coefficients of five proteins (lysozyme, chymotrypsinogen-A, bovine serum albumin, transferrin and catalase) in 16 sets of polyethylene glycol (PEG)/dextran systems were correlated. In addition, partition behavior of lactate dehydrogenase in PEG/hydroxypropyl starch systems were measured, correlated and partially predicted. A comparison of calculated and experimental data indicated that the model provides good correlation and prediction abilities on the protein partitioning in aqueous polymer two-phase systems with a wide range of polymer concentration and molecular weight.     

Aqueous two‐phase micellar systems in an oscillatory flow micro‐reactor: study of perspectives and experimental performance

BACKGROUND: Aqueous two‐phase micellar systems (ATPMS) are micellar surfactant solutions with physical properties that make them very efficient for the extraction/concentration of biological products. In this work the main proposal that has been discussed is the possible applicability and importance of a novel oscillatory flow micro‐reactor (micro‐OFR) envisaged for parallel screening and/or development of industrial bioprocesses in ATPMS. Based on the technology of oscillatory flow mixing (OFM), this batch or continuous micro‐reactor has been presented as a new small‐scale alternative for biological or physical‐chemical applications. RESULTS: ATPMS experiments were carried out in different OFM conditions (times, temperatures, oscillation frequencies and amplitudes) for the extraction of glucose‐6‐phosphate dehydrogenase (G6PD) in Triton X‐114/buffer with Cibacron Blue as affinity ligand. CONCLUSION: The results suggest the potential use of OFR, considering this process a promising and new alternative for the purification or pre‐concentration of bioproducts. Despite the applied homogenization and extraction conditions have presented no improvements in the partitioning selectivity of the target enzyme, when at rest temperature they have influenced the partitioning behavior in Triton X‐114 ATPMS. Copyright © 2011 Society of Chemical Industry     

Improved recovery of B‐phycoerythrin produced by the red microalga Porphyridium cruentum

A simplified process for the primary recovery and purification of B‐phycoerythrin (BPE) from Porphyridium cruentum exploiting aqueous two‐phase systems (ATPS) and isoelectric precipitation was developed in order to reduce the number of unit operations and benefit from increased purity and yield of the protein product. Evaluation of the partitioning behaviour of BPE in polyethylene glycol (PEG)/sulphate, PEG/dextran and PEG/phosphate ATPS was carried out to determine under what conditions the BPE and contaminants concentrated into opposite phases. An additional stage of isoelectric precipitation at pH 4.0 after cell disruption resulted in an increase in purity of the target protein from the BPE crude extract and enhanced the performance of the subsequent ATPS. PEG1000/phosphate ATPS proved to be suitable after isoelectric precipitation for the recovery of highly purified (defined as absorbance ratio A_{545 nm}/A_{280 nm} > 4.0) BPE with a potential commercial value as high as US$ 50/mg. An ATPS extraction stage comprising 29.5% (w/w) PEG1000, 9.0% (w/w) phosphate, a volume ratio (V_r) equal to 1.0, a system pH of 7.0 and loaded with 40% (w/w) of the BPE extract generated by precipitation allowed BPE recovery with a purity of 4.1±0.2 and an overall product yield of 72% (w/w). The purity of BPE from the crude extract increased 5.9‐fold after isoelectric precipitation and ATPS. The results reported herein demonstrate the benefits of the practical application of isoelectric precipitation together with ATPS for the recovery and purification of BPE produced by P. cruentum as a first step in the development of a commercial purification process. Copyright © 2006 Society of Chemical Industry