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.     

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 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%.     

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.     

Separation of catalase from Amsonia orientalis with single step by aqueous two-phase partitioning system (ATPS)

Catalase from Amsonia orientalis was purified by ATPS, and its efficiency was compared against hydrophobic interaction chromatography. Activity recovery and purification fold of purified catalase by ATPS were examined under varying experimental conditions. The effects of various factors such as type of phase-forming salts, (PEG) mass, with their different concentrations, pH and temperature effects on partitioning were investigated. The highest activity recovery (156%) and purification fold (8.67) of catalase were obtained in the ATPS system containing 10% (g/g) PEG4000, 15% (g/g) Na₂SO₄ at pH 6.0 and room temperature. In hydrophobic interaction chromatography, the enzyme has been purified 12.54-fold with 57.5% recovery. The molecular weight of catalase was determined as 75 kDa by SDS-PAGE.     

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     

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     

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.     

Extractive bioconversion of xylan for production of xylobiose and xylotriose using a PEG6000/sodium citrate aqueous two-phase system

Aqueous two-phase system (ATPS) was applied for extraction bioconversion of xylan by xylanase from Trichoderma viride. Phase diagrams for poly (ethylene glycol) (PEG) and sodium citrate were determined at room temperature. The ATPS composed of 12.99% (w/w) PEG6000 and 12.09% (w/w) sodium citrate was favorable for partition of xylanase and used for extraction bioconversion of xylan. Batch hydrolysis demonstrated that higher concentrations of xylobiose and xylotriose were obtained in the PEG6000/sodium citrate ATPS compared to those in the aqueous system. These results present the potential feasibility of production of xylo-oligosaccharides by extraction bioconversion in ATPS.     

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.     

双水相体系萃取精氨酸脱亚胺酶

报道了利用聚乙二醇/硫酸铵双水相体系从自溶NJ402菌粗提酶液中分离纯化精氨酸脱亚氨酶(ADI)的研究结果,为精氨酸脱亚氨酶的分离纯化提供了一种方法。在双水相体系中采用聚乙二醇(PEG)与(NH4)2SO4为组成成分,考察了聚乙二醇(PEG)平均相对分子质量、PEG质量分数、(NH4)2SO4质量分数、pH及NaCl质量分数对精氨酸脱亚氨酶分离纯化效果的影响。最佳双水相体系萃取条件为:聚乙二醇(PEG)平均相对分子质量为1 000,w(PEG1000)=15%,w[(NH4)2SO4]=20%,pH=6.5,室温下从自溶NJ402菌粗提酶液中分离纯化精氨酸脱亚氨酶,纯化倍数达到2.35倍,萃取率达91.1%。     

New aqueous two‐phase systems based on poly(ethylene oxide sulfide) (PEOS) and potassium phosphate for the potential recovery of proteins

A new aqueous two‐phase system (ATPS) based on a degradable polymer called poly(ethylene oxide sulfide) with a molecular weight of 33 000 g mol^?1 (identified as PEOS‐12) and potassium phosphate was exploited for the potential recovery of proteins. An initial characterisation of the ATPS was achieved by the construction of a phase diagram for the PEOS‐12/phosphate system. The protein partitioning behaviour of lysozyme and bovine serum albumin (BSA), selected as single model proteins, and B‐phycoerythrin (BPE) produced by Porphyridium cruentum in the new ATPS under increasing tie line length (TLL) conditions at constant phase volume ratio (V_r) and system pH was investigated. Both single proteins partitioned in the new ATPS, initially exhibiting bottom phase preference; however, lysozyme changed phase preference when TLL was increased. Fractionation of a complex model (production of BPE by P. cruentum) using PEOS‐12/phosphate ATPS was performed to evaluate the potential protein recovery from fermentation broth or cell homogenate. The proposed new ATPS proved to be suitable for the potential recovery of BPE from crude extract of P. cruentum. In general, a system comprising V_r = 1.0, 18% (w/w) PEOS‐12, 8% (w/w) phosphate and 30% (w/w) TLL at pH 7.0 provided conditions to concentrate BPE into the bottom phase (i.e. partitioning behaviour of BPE; lnK_BPE = ?1.8) with a protein recovery of 84%. The findings reported here demonstrate the potential application of the new ATPS for the recovery of proteins from complex biological suspensions. Copyright © 2006 Society of Chemical Industry     

Application of Aqueous Two‐Phase Systems for the Potential Extractive Fermentation of Cyanobacterial Products

The potential use of aqueous two‐phase systems (ATPS) to establish a viable protocol for the in situ recovery of cyanobacterial products was evaluated. The evaluation of system parameters such as poly (ethylene glycol) (PEG) molecular mass, concentration of PEG and salt was carried out to determine the conditions under which Synechocystis sp. PCC 6803 cell and cyanobacterial products, i.e., β‐carotene and lutein, become concentrated in opposite phases. PEG‐phosphate ATPS proved to be unsuitable for the recovery of cyanobacterial products due to the negative effect of the salt upon the cell growth. The use of ATPS PEG‐dextran (6.6 % w/w PEG 3350, 8.4 % w/w dextran 66900, TLL 17.3 % w/w, V_R 1.0, pH 7) and (4.22 % w/w PEG 8000, 9.77 % w/w dextran 66900, TLL 18 % w/w, V_R 1.0, pH 7) resulted in the growth of cyanobacteria (Synechocystis sp. PCC 6803) and the concentration of lutein in opposite phases. However, β‐carotene was seen to concentrate in the top phase together with the biomass. The results reported here demonstrate the potential application of ATPS to establish the conditions for an extractive fermentation prototype process for the recovery of cyanobacterial products.     

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％。     

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     

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.     

Extracellular endo-mannanase from Bacillus sp. CFR1601: Economical production using response surface methodology and downstream processing using aqueous two phase system

Bacillus sp. CFR1601, isolated from decaying plant litter, produced an extra-cellular endo-mannanase (198.0 IU/g) under solid state fermentation (SSF) using defatted coconut residue as the prime solid substrate. In order to enhance endo-mannanase production, three component, five level central composite design (CCD) of response surface methodology (RSM) was used. Based on contour plots and variance analysis, optimum conditions for endo-mannanase production from Bacillus sp. CFR1601 were attained when defatted coconut residue was supplemented with sesame oil meal (10.0, w/w), Tween-80 (0.2%, v/v) and inoculated with bacterial cells from log phase (12 h old; OD_600 nm ≈ 3.6). The empirical model developed through RSM brought about 4.04–4.39-fold (800.0–870.0 IU/g) improvement in endo-mannanase yield as compared to un-optimized growth conditions. Downstream processing of endo-mannanase from SSF media was carried out for the first time using polyethylene glycol (PEG)/salt aqueous two phase system (ATPS). ATPS system consisting of a combination of PEG 3350 12.0% (w/w), Na₂SO₄ 12.0% (w/w), protein load 10.0% (w/w) and pH 5.0 resulted in one-sided partitioning of endo-mannanase towards bottom phase with 3.8-fold purification and 95.4% recovery. Second stage ATPS with fresh top phase further improved purification of endo-mannanase to 12.32-fold. Our overall results suggest a cost-effective and integrated process for production and downstream processing of endo-mannanase.     

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.     

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.