Separation of Bromelain by Aqueous Two Phase Flotation

Aqueous two phase flotation (ATPF) system of polyethylene glycol (PEG) and potassium phosphate is studied for the separation and partial purification of bromelain from the pineapple fruit (Annanus comosus L. Merryl). The effect of PEG molecular weight (1500–20000), concentration of phase forming components (PEG 12–18% w/w and potassium phosphate 14–20% w/w), system pH, nitrogen flow rate, and flotation time were studied and optimum conditions for ATPF were obtained. At optimum conditions of the system, i.e., 14% w/w PEG 1500, 18% w/w potassium phosphate, 80 mL/min of nitrogen flow rate and pH 7, maximum yield of 91.47% and purification fold of 4.26 were obtained. ATPF was found to be an effective technique for the purification of bromelain from pineapple fruit with higher extraction yield and purification fold as compared to aqueous two phase extraction (ATPE).     

The optimization of aqueous two‐phase extraction of lysozyme from crude hen egg white using response surface methodology

BACKGROUND: Aqueous two‐phase extraction is a versatile method for separating biological particles and macromolecules. In the present wok, the feasibility of using PEG 4000/potassium citrate aqueous two‐phase system (ATPS) for recovering and purifying lysozyme was investigated. Response surface methodology was used to determine an optimized ATPS for purification of lysozyme from crude hen egg white. RESULTS: Mathematical models concerning the purification of lysozyme from chicken egg white in polyethylene glycol 4000 (PEG 4000)/potassium citrate ATPS are established using response surface methodology. Screening experiments using fractional factorial designs show that the pH of the system significantly affects the recovery and purification of lysozyme. An optimized ATPS was proved to be at pH 5.5 and 30 °C and contained 18% (w/w) PEG, 16% (w/w) potassium citrate, 3.75% (w/w) potassium chloride (KCl). Under those conditions, the specific activity, purification factor and activity yield for lysozyme were 31100 U mg^?1, 21.11 and 103%, respectively. CONCLUSION: The PEG 4000/potassium citrate ATPS has the potential to be applied to establish bioprocesses for the primary recovery and partial purification of lysozyme. © 2012 Society of Chemical Industry     

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.     

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.     

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.     

A simple method for purification of bromelain in a thermosensitive triblock copolymer-based protection system and recycling of phase components

In the work, we chose stem bromelain as a model to investigate the storage and purification of bromelain from pineapple peel. Extraction of bromelain from pineapple peel using a two-stage aqueous two-phase extraction system composed of a thermoseparating copolymer EOPOEO and K₂HPO₄. Bromelain predominantly partitioned to the EOPOEO-rich phase and then re-extract to the top dilute phase. The recovery of enzyme activity (68.6%) and purification factor (6.53) were determined under optimum conditions. The EOPOEO-rich phase and salt were recycled, and the recovery of enzyme activity could reach up to 60%. This method has been proved to obtain highly purified and stable bromelain.     

Impact of polyethylene glycol as additive on the formation and extraction behavior of ionic‐liquid based aqueous two‐phase system

Developing a novel Ionic‐liquid (IL) based aqueous two‐phase system (ATPS) with polyethylene glycol (PEG) as adjuvant for the separation of biomolecules is studied. This original work involves addition of various concentration of PEG (2000, 4000, and 6000 gr/mol) to 1‐butyl‐3‐methylimidazolium acetate+ potassium hydrogen phosphate ATPS to investigate their subsequent effect on phase diagrams and partitioning coefficient of α‐amylase. In another innovative aspect of this work, response surface methodology (RSM) based on three‐variable central composite design was employed to understand the effect of phase forming components on extraction studies of α‐amylase. The addition of small amount of PEG improved the partitioning coefficient of biomolecule. The effective excluded volume theory was applied to correlate the salting‐out ability. As a result, it can be stated that the proposed system can effectively be used in separation and purification studies instead of task specific ILs. © 2015 American Institute of Chemical Engineers AIChE J, 62: 264–274, 2016     

Improving glutathione extraction from crude yeast extracts by optimizing aqueous two-phase system composition and operation conditions

PEG-Dextran and PEG-salt aqueous two-phase systems (ATPS) have been applied to separate glutathione (GSH) from crude yeast extracts. Single-factor experiments were carried out to determine the important factors influencing the partition coefficient and extraction yield. The effect of PEG molecular weight, phase-forming components, PEG and Dextran concentration, pH value, and temperature on the GSH partitioning behavior in ATPS was investigated. Three factors, Dextran concentration, pH value, and temperature, were confirmed to have significant influence on the partition coefficient and extraction yield. These factors were further analyzed with the aid of central composite rotatable design and response surface methodology. The optimal conditions for GSH extraction in the PEGDextran system were determined, including PEG molecular weight 6,000, 10% PEG concentration, 14% Dextran concentration, pH 5.2, and temperature 32 °C. A high extraction yield (83.55%) of GSH from crude yeast extracts was achieved under these optimized conditions. This work is very helpful for developing one efficient and cost-effective process for the separation and purification of GSH from yeast broths.     

Optimization of Rhizopus niveus Lipase Partitioning by an Aqueous Biphasic System

An unconventional aqueous two‐phase system (ATPS) composed of polyethylene glycol (PEG) and sodium carbonate was developed and optimized by employing response surface methodology for separation of Rhizopus niveus lipase. A five‐level central composite design was applied to evaluate the optimal level of three process variables in order to obtain maximum lipase separation. Experimental data were analyzed by regression and a polynomial model was created which was found significant. The maximum partition coefficient was achieved with the system PEG 4000/sodium carbonate. Validation experiments confirmed the high accordance of predicted and experimental results. The optimized ATPS can be applied as a suitable cost‐effective system for lipase extraction.     

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     

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.     

Potential application of aqueous two‐phase systems for the fractionation of RNase A and α‐Lactalbumin from their PEGylated conjugates

BACKGROUND: PEGylation reactions often result in a heterogeneous population of conjugated species and unmodified proteins that presents a protein separations challenge. Aqueous two‐phase systems (ATPS) are an attractive alternative for the potential fractionation of native proteins from their PEGylated conjugates. The present study characterizes the partition behaviors of native RNase A and α‐Lac and their mono and di‐PEGylated conjugates on polyethylene glycol (PEG)—potassium phosphate ATPS. RESULTS: A potential strategy to separate unreacted native protein from its PEGylated species was established based upon the partition behavior of the species. The effect of PEG molecular weight (400–8000 g mol^?1), tie‐line length (15–45% w/w) and volume ratio (V_R; 0.33, 1.00 and 3.00) on native and PEGylated proteins partition behavior was studied. The use of ATPS constructed with high PEG molecular weight (8000 g mol^?1), tie‐line lengths of 25 and 35% w/w, and V_R values of 1.0 and 3.0 allowed the selective fractionation of native RNase A and α‐Lactalbumin, respectively, from their PEGylated conjugates on opposite phases. Such conditions resulted in an RNase A bottom phase recovery of 99%, while 98% and 88% of mono and di‐PEGylated conjugates, respectively were recovered at the top phase. For its part, α‐Lac had a bottom phase recovery of 92% while its mono and di‐PEGylated conjugates were recovered at the top phase with yields of 77% and 76%, respectively. CONCLUSIONS: The results reported here demonstrate the potential application of ATPS for the fractionation of PEGylated conjugates from their unreacted precursors. Copyright © 2010 Society of Chemical Industry     

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.     

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.     

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.     

Aqueous two-phase countercurrent distribution for the separation of c-phycocyanin and allophycocyanin from Spirulina platensis

C-phycocyanin (C-PC) and allophycocyanin (APC) with similar molecular structures were separated, respectively from Spirulina platensis cell homogenate by single extraction and multi-stage countercurrent distribution (CCD) using an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and potassium phosphate (KPi). The partition coefficients of C-PC and APC were 10.64 and 0.57, respectively, and the extraction selectivity of C-PC was 18.67 from 0.5% (w/w) S. platensis crude extract by single extraction using PEG6000/KPi ATPS (pH 7.0) with 34% (w/w) tie line length (TLL). In ten-stage CCD under the same ATPS extraction condition with 2% (w/w) S. platensis crude extract, the purity of C-PC increased nearly twice and the recovery of APC increased more than nine-fold compared with single extraction. The results displayed that most C-PC (82.1%) followed the mobile phase was enriched in the top phases of the last three tubes, while more APC (41%) remained in the stationary phase was enriched in the bottom phases of the first three tubes in the ten-stage CCD. Hence, aqueous two-phase CCD technology provided an effective and low cost method for C-PC and APC separation from S. platensis cell homogenate directly.     

Partitioning of lysozyme in aqueous two-phase systems containing ethylene oxide-propylene oxide copolymer and potassium phosphates

Lysozyme partitioning in EO₅₀PO₅₀/potassium phosphate aqueous two-phase systems (ATPS) was studied. In the work, the influence of EO₅₀PO₅₀, potassium phosphate and sodium chloride concentration in the ATPS on lysozyme partition coefficient and separation yield was examined. In addition, the influence of the pH of potassium phosphate solution was also investigated. A Box–Behnken design was defined, and response surface models for the partition coefficient K and percentage yield of the enzyme in the top phase Y were calculated. Among the examined factors, the NaCl concentration had the highest influence on lysozyme separation parameters. This influence can be explained mainly by the hydrophobic interactions between the protein and the phase-forming components. A maximum partition coefficient K_L1, yield Y_L1 and Y_L2 were predicted for EO₅₀PO₅₀, potassium phosphate and NaCl concentrations of 17.40, 22.67% and 0.85 mol/l, respectively, and for pH 9.0. A good agreement was obtained between the experimental and the predicted results.     

Practical application of aqueous two‐phase systems for the development of a prototype process for c‐phycocyanin recovery from Spirulina maxima

A novel process for the recovery of c‐phycocyanin from Spirulina maxima exploiting aqueous two‐phase systems (ATPS), ultrafiltration and precipitation was developed in order to reduce the number of unit operations and benefit from an increased yield of the protein product. The evaluation of system parameters such as PEG molecular mass, concentration of PEG as well as salt, system pH and volume ratio was carried out to determine under which conditions the c‐phycocyanin and contaminants concentrate to opposite phases. PEG1450–phosphate ATPS proved to be suitable for the recovery of c‐phycocyanin because the target protein concentrated in the top phase whilst the cell debris concentrated in the bottom phase. A two‐stage ATPS process with a phase volume ratio (V_r) equal to 0.3, PEG1450 7% (w/w), phosphate 20% (w/w) and system pH of 6.5 allowed c‐phycocyanin recovery with a purity of 2.4 (estimated as the relationship of the 620 nm to 280 nm absorbances). The use of ultrafiltration (with a 30 kDa membrane cut‐off) and precipitation (with ammonium sulfate) resulted in a recovery process that produced a protein purity of 3.8 ± 0.1 and an overall product yield of 29.5% (w/w). The results reported here demonstrated the practical implementation of ATPS for the design of a prototype recovery process as a first step for the commercial purification of c‐phycocyanin produced by Spirulina maxima. © 2001 Society of Chemical Industry     

Reverse micellar extraction of bromelain from Ananas comosus L. Merryl

BACKGROUND: A reverse micellar system (RMS) of ionic surfactants is used for the first time for the extraction and primary purification of fruit bromelain (EC 3.4.22.33) from the aqueous extract of pineapple (Ananas comosus L. Merryl). The effect of various process parameters on both forward and back extraction of bromelain is studied to improve the extraction efficiency of RMS. Most of the reverse micellar extraction (RME) studies reported so far are on model systems and its application to enzyme extraction from a natural source is rarely reported. RESULTS: Studies carried out with ionic surfactants sodium bis(2‐ethylhexyl)sulfosuccinate (AOT) and cetyltrimethylammonium bromide (CTAB) confirmed that electrostatic interaction was the main driving force for the extraction of fruit bromelain. Among the two surfactants studied, CTAB was found to be the most suitable for the extraction of fruit bromelain with respect to activity recovery (97.56%) and degree of purification (4.54 fold) when employed as a 150 mmol L^?1 CTAB/iso‐octane/5% (v/v) hexanol/15% (v/v) butanol system. Activity recovery with a counterionic system is higher (94.30%) in comparison with isopropyl alcohol added system (85.35%). CONCLUSION: RME could be used as an efficient primary purification step for the recovery of bromelain from pineapple juice. Reverse micellar phase components can easily be recovered and efficiently reused for fresh or subsequent extraction, which contributes favorably to the process economics and environmental issues. Copyright © 2007 Society of Chemical Industry     

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％。