Integration of production and aqueous two-phase systems extraction of extracellular Fusarium solani pisi cutinase fusion proteins

Genetic engineering was integrated with the production and purification of Fusarium solani pisi cutinases, in order to obtain the highest amount of enzyme activity units, after purification. An aqueous two-phase system (ATPS) of polyethylene glycol 3350, dipotassium phosphate and whole broth was used for the extraction of three extracellular cutinases expressed in Saccharomyces cerevisiae. The production/extraction process was evaluated regarding cutinases secretion in the medium, partition behaviour and extraction yields in the ATPS. The proteins studied were cutinase wild type and two fusion proteins of cutinase with the tryptophane-proline (WP) fusion tags, namely (WP)(2) and (WP)(4). The (WP)(4) fusion protein enabled a 300-fold increase of the cutinase partition coefficient when comparing to the wild type. However, the secretion of the fusion proteins was lower than of the wild type cutinase secretion. A batch extraction strategy was compared with a continuous extraction in a perforated rotating disc contactor (PRDC). The batch and continuous systems were loaded with as much as 60% (w/w) whole cultivation broth. The continuous extraction strategy provided a 2.5 higher separation capacity than the batch extraction strategy. Considering the integrated process, the cutinase-(WP)(2) proved to lead to the highest product activity, enabling five and six times more product activity than the wild type and the (WP)(4) fusion proteins, respectively.     

Extraction of peptide tagged cutinase in detergent-based aqueous two-phase systems

Detergent-based aqueous two-phase systems have the advantage to require only one auxiliary chemical to induce phase separation above the cloud point. In a systematic study the efficiency of tryptophan-rich peptide tags was investigated to enhance the partitioning of an enzyme to the detergent-rich phase using cutinase as an example. Up to 90% enzyme activity could be extracted in a single step from whole broth of recombinant Saccharomyces cerevisiae expressing cutinase variants carrying a (WP)₄ tag. In contrast, the extraction yield of wild type cutinase was 2–3% only. The detergent concentration and the temperature are the main parameters to optimize the extraction yield. Considering availability, extraction yields, and price the detergent Agrimul NRE 1205 served best for enzyme recovery.     

Extractive disruption process integration using ultrasonication and an aqueous two‐phase system for protein recovery from Chlorella sorokiniana

Microalgae emerge as the most promising protein sources for aquaculture industry. However, the commercial proteins production at low cost remains a challenge. The process of harnessing microalgal proteins involves several steps such as cell disruption, isolation and extraction. The discrete processes are generally complicated, time‐consuming and costly. To date, the notion of integrating microalgal cell disruption and proteins recovery process into one step is yet to explore. Hence, this study aimed to investigate the feasibility of applying methanol/potassium ATPS in the integrated process for proteins recovery from Chlorella sorokiniana. Parameters such as salt types, salt concentrations, methanol concentrations, NaCl addition were optimized. The possibility of upscaling and the effectiveness of recycling the phase components were also studied. The results showed that ATPS formed by 30% (w/w) K₃PO₄ and 20% (w/w) methanol with 3% (w/w) NaCl addition was optimum for proteins recovery. In this system, the partition coefficient and yield were 7.28 and 84.23%, respectively. There were no significant differences in the partition coefficient and yield when the integrated process was upscaled to 100‐fold. The recovered phase components can still be recycled effectively at fifth cycle. In conclusions, this method is simple, rapid, environmental friendly and could be implemented at large scale.     

Potential Aqueous Two‐Phase Processes for the Primary Recovery of Colored Protein from Microbial Origin

The primary recovery of c‐phycocyanin and b‐phycoerythrin from Spirulina maxima and Porphyridium cruentum, respectively, using an established extraction strategy was selected as a practical model system to study the generic application of polyethylene glycol (PEG)‐phosphate aqueous two‐phase systems (ATPS). The generic practical implementation of ATPS extraction was evaluated for the recovery of colored proteins from microbial origin. A comparison of the influence of system parameters, such as PEG molecular mass, concentration of PEG as well as salt, system pH and volume ratio, on the partition behavior of c‐phycocyanin and b‐phycoerythrin was carried out to determine under which conditions target colored protein and contaminants concentrate to opposite phases. One‐stage processes are proposed for the primary recovery of the colored proteins. PEG1450‐phosphate ATPS extraction (volume ratio (V_R) equal to 0.3, tie‐line length (TLL) of 34 % w/w and system pH 7.0) for the recovery of c‐phycocyanin from Spirulina maxima resulted in a primary recovery process that produced a protein purity of 2.1 ± 0.2 (defined as the relationship of 620 nm to 280 nm absorbance) and a product yield of 98 % [w/w]. PEG1000‐phosphate ATPS extraction (i.e., V_R = 1.0, PEG 1000, TLL 50 % w/w and system pH 7.0) was preferred for the recovery of b‐phycoerythrin from Porphyridium cruentum, which resulted in a protein purity of 2.8 ± 0.2 (defined as the relationship of 545 nm to 280 nm absorbance) and a product yield of 82 % [w/w]. The purity of c‐phycocyanin and b‐phycoerythrin from the crude extract increased 3‐ and 4‐fold, respectively, after ATPS. The results reported herein demonstrated the benefits of the practical generic application of ATPS for the primary recovery of colored proteins from microbial origin as a first step for the development of purification processes.     

Extractive cultivation of Lactococcus lactis using a polyethylene glycol/MgSO4 · 7H2O aqueous two-phase system to produce nisin

To relieve lactic acid inhibition, an aqueous two-phase system (ATPS) was used to grow Lactococcus lactis. Its composition was 11% (w/v) PEG 20000/3.5% (w/v) MgSO₄ 7H₂O. In this ATPS medium, the cells were completely partitioned in the bottom phase, and lactic acid had the biggest partition coefficient of the eight ATPS media tested. The cell biomass in this medium was 0.64 mg ml^–1, only 60% of that of the control medium, but nisin production (803 IU ml^–1) was enhanced by 33%. The increase in nisin was explained as a result of extraction of lactic acid from the bottom phase to the top one. The changes of tie-line length and phase volume ratio for the identical tie line could affect cell growth and nisin accumulation.     

Pilot-scale extraction of an intracellular recombinant cutinase from E. coli cell homogenate using a thermoseparating aqueous two-phase system

A thermoseparating aqueous two-phase system for extraction of a recombinant cutinase fusion protein from Escherichia coli homogenate has been scaled up to pilot scale. The target protein ZZ-cutinase-(WP)(4) was produced in a fed batch process at 500 l to a concentration of 12% of the total protein and at a cell concentration of 19.7 g l(-1). After harvest and high-pressure homogenisation a first extraction step was performed in an EO(50)PO(50) (50% (w/w) ethylene oxide and 50% (w/w) propylene oxide) thermopolymer/amylopectin rich Waxy barley starch system. The (WP)(4) tag was used for enhanced target protein partitioning to the EO(50)PO(50) phase while the cell debris was collected in the starch phase. A second extraction step followed where the recovered EO(50)PO(50) phase from the first step was supplemented with a non-ionic detergent (C(12-18)EO(5)) and heated to the cloud point (CP) temperature (45 degrees C). One polymer-rich liquid phase and one almost pure aqueous phase were formed. The target protein could be obtained in a water phase after the thermal phase separation at a total recovery over the extraction steps of 71% and a purification factor of 2.5. We were able to demonstrate that a disk-stack centrifugal separator could be adapted for rapid separation of both primary and thermoseparated phase systems.     

Purification and Characterization of Polyphenol Oxidase From Waste Potato Peel by Aqueous Two-Phase Extraction

Potato peel from food industrial waste is a good source of polyphenol oxidase (PPO). This work illustrates the application of an aqueous two-phase system (ATPS) for the extraction and purification of PPO from potato peel. ATPS was composed of polyethylene glycol (PEG) and potassium phosphate buffer. Effect of different process parameters, namely, PEG, potassium phosphate buffer, NaCl concentration, and pH of the system, on partition coefficient, purification factor, and yield of PPO enzyme were evaluated. Response surface methodology (RSM) was utilized as a statistical tool for the optimization of ATPS. Optimized experimental conditions were found to be PEG1500 17.62% (w/w), potassium phosphate buffer 15.11% (w/w), and NaCl 2.08 mM at pH 7. At optimized condition, maximum partition coefficient, purification factor, and yield were found to be 3.7, 4.5, and 77.8%, respectively. After partial purification of PPO from ATPS, further purification was done by gel chromatography where its purity was increased up to 12.6-fold. The purified PPO enzyme was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by K_m value 3.3 mM, and V_max value 3333 U/mL, and enzyme stable ranges for temperature and pH of PPO were determined. These results revealed that ATPS would be an attractive option for obtaining purified PPO from waste potato peel.     

Application of TMA-PEG to promote C-phycocyanin extraction from S. platensis in the PEG ATPS

A novel aqueous two phase system (ATPS) using trimethylamine-polyethylene glycols (TMA-PEG) to promote the extraction of C-phycocyanin (C-PC) from S.platensis was introduced. The purity of C-PC (EP) obtained in the ATPS of PEG1000/Na₃PO₄ was increased 2.1 times by the addition of TMA-PEG1000. The purification factor was enhanced from 2.9 to 10.1 when 65% TMA-PEG1000 was added in the system. The ATPS operation must be carried out in the pH range of 6.0-7.0 and at temperatures less than 35 °C for maintaining the stability of C-PC. The partition coefficient and recovery ratio of C-PC increased with the increasing concentration of TMA-PEG. The system parameters like TMA-PEG1000 content, tie line length (TLL), pH, temperature and phase volume ratio (V_r) were screened and optimized using the fractional factorial design and Box-Behnken experiment design. The optimized system is composed of 11.8% PEG1000/TMA-PEG1000 (w/w), 64.42% TMA-PEG1000 (w/w PEG1000) and 9.5% Na₃PO₄ (w/w) with 38.19% TLL (w/w) and 0.89 V_r at pH 6.5 and 25 °C. The obtained value of EP was 5.21 in one-stage ATPS and 6.7 in two-stage ATPS. The recovery ratio of C-PC in the new ATPS extraction system was more than 97%.     

Purification and characterization of cutinase from Bacillus sp. KY0701 isolated from plastic wastes

A total of approximately 400 bacterial strains were isolated from 73 plastic wastes collected from 14 different regions. Nineteen isolates that form clear zones both on tributyrin and poly ε-caprolactone (PCL) agar, were identified based on 16S rRNA gene sequences. Among these, Bacillus sp. KY0701 that caused the highest weight loss of PCL films in minimal salt medium, was selected for cutinase production. The highest enzyme activity (15 U/mL) was obtained after 4 days of incubation at 50°C, pH 7.0 and 200?rpm in a liquid medium containing 1.5% (w/v) apple cutin and 0.1% (w/v) yeast extract. The purified enzyme was stable at high temperatures (50–70°C) and over a wide pH range (5.5–9.0). The relative activity of cutinase was at least 75% in the percent of various organic solvents. The apparent K_m and V_max values of the cutinase for p-nitrophenyl butyrate were 0.72?mM and 336.8?µmol p-nitrophenol/h/g, respectively. In addition, it showed high stability and compatibility with commercial detergents. These features of cutinase obtained from Bacillus sp. KY0701 make it a promising candidate for application in the detergent and chemical industries. In our best knowledge, this is the first report for cutinase production and characterization produced by a Bacillus strain.     

Optimization of conditions for acid protease partitioning and purification in aqueous two-phase systems using response surface methodology

Aspergillopepsin I, an acid protease, was purified using an aqueous two-phase system that comprised various combinations of polyethylene glycol (PEG), NaH₂PO₄ and NaCl. Partition of the enzyme depended upon the molecular mass of the PEG and the presence of NaCl. With PEG 1500, 4000 and 6000, the partition coefficients were increased by 1,500-, 1,800- and 560-fold compared to values without NaCl. The presence of NaCl (8.75%, w/w) increased purification by 3.8, 9.5 and 2.8 times into these respective PEGs. The optimal aqueous two-phase system for acid protease purification was developed using response surface methodology. This system contained 17.3% of PEG 4000 (w/w), 15% NaH₂PO₄ (w/w) and 8.75% NaCl (w/w) and provided the best partition coefficient (Ke > 1,100) and yield over 99% in the same phase. The optimal ATPS purification factor of acid protease was over 5.     

Cutinase purification on poly(ethylene glycol)–hydroxypropyl starch aqueous two-phase systems

The partition behaviour of cutinase on poly(ethylene glycol) (PEG)–hydroxypropyl starch aqueous two-phase systems was characterized. The effect of molecular mass of PEG, the pH of the system and tie-line length on cutinase partition coefficient and cutinase yield to the top phase was investigated for systems prepared with a purified hydroxypropyl starch (Reppal PES 100) and a crude one (HPS). The effect of the presence of different salts, such as sodium chloride, sodium sulphate and ammonium sulphate, on cutinase partition was also studied. The results lead to the conclusion that aqueous two-phase systems composed of PEG and hydroxypropyl starch are not efficient in the purification of cutinase. In the majority of cases, the partition coefficients were very close to 1, with pH being the factor which affects most cutinase partition. Partition coefficients were significantly improved when salts were added to the systems. For PEG 4000–Reppal PES 100 [at pH 4.0; 0.5 M (NH₄)₂SO₄], the partition coefficient for cutinase was 3.7, while a value of 12 was obtained for PEG 4000–HPS (at pH 4.0; 1 M NaCl). An isoelectric point (pI) of 7.8 was confirmed for cutinase by constructing a cross partition graphic from the results obtained in the experiments with different salts.     

Production,extraction, and thermodynamics protease partitioning from Aspergillus tamarii Kita UCP1279 using PEG/sodium citrate aqueous two-phase systems

Abstract

The protease from Aspergillus tamarii Kita UCP1279 extraction by aqueous two-phase PEG-Citrate (ATPS) systems, using a factorial design 2⁴, was investigated. Then, the variables studied were polyethylene glycol (PEG) molar mass (M_PEG), concentrations of PEG (C_PEG) and citrate (C_CIT), and pH. The responses analyzed were the partition coefficient (K), activity yield (Y) and purification factor (PF). The thermodynamic parameters of the ATPS partition were estimated as a function of temperature. ATPS was able to pre-purify the protease (PF = 1.6) and obtained 84% activity yield. The thermodynamic parameters ΔG°_m (?10.89?kJ mol^?1), ΔH_m (?5.0?kJ?mol^?1) and partition ΔS_m (19.74?J mol^?1 K^?1) showed that the preferential migration of almost all protein contaminants of the crude extract to the salt-rich phase, while the preferred protease was the PEG rich phase. The extracted enzyme presents optimum temperature and pH at range of 40–50?°C and 9.0–11.0, respectively. Moreover, the enzyme was identified as serine protease based on inhibition profile. ATPS showed the satisfactory performance as the first step for Aspergillus tamarii Kita UCP1279 protease pre-purification.     

AQUEOUS TWO-PHASE EXTRACTION FOR THE PURIFICATION OF ALKALINE AGARASES FROM CULTURE EXTRACTS OF Pseudomonas aeruginosa AG LSL-11

The agarases were purified for the first time an using aqueous two-phase system (ATPS) consisting of polyethylene glycol (PEG) and phosphate salt. The three extracellular, alkaline agarases produced by Pseudomonas aeruginosa AG LSL-11 were efficiently extracted into the top PEG-rich layer. The influencing factors on the partition of agarases—molecular weight of the PEG, system pH, system temperature, and NaCl concentration—were investigated. All the factors were found to have a significant effect on the partition of agarases except NaCl. The optimal ATPS parameters for the partitioning and purification of agarases were found to be 12% PEG 600 and 11.9% (w/w) phosphate salt at pH 8.0 and 4°C. All three agarases were concentrated in the top PEG phase with 6.19-fold purity and 71.21% recovery. The ATPS was found to be more convenient and economical than the conventional ion-exchange chromatography (IEC) method for extraction of three agarases and could be significantly employed for the purification of agarases from fermentation broth.     

Separation and determination of tetracycline hydrochloride in real water samples using binary small molecule alcohol‐salt aqueous two‐phase system coupled with high‐performance liquid chromatography

An environmental and sensitive sample pretreatment method was established and combined with high‐performance liquid chromatography (HPLC) for the analysis of tetracycline hydrochloride (TC) in feed water and lake water. One element small molecule alcohol‐salt aqueous two‐phase system (ATPS) cannot effectively adjust the polarity of the system, but binary small molecule alcohol‐salt ATPS can adjust the polarity and improve the extraction efficiency of antibiotics. In this work, a binary ATPS based on ethanol +2‐propanol + (NH₄)₂SO₄ system was formed and applied to the separation and purification of TC in real water samples. The influence factors on partition behaviors of TC were discussed, including the types and the concentration of phase salts, the volume ratio of alcohol, the pH value, extraction temperature, and the standing time. The response surface methodology was used to determine the best experimental conditions for multi‐factor experiments. Under this optimal condition, the extraction efficiency of TC reached 95.7%. This new method is considered to have significant application in the divorce of antibiotics.     

Investigation of chromatography and polymer/salt aqueous two-phase processes for downstream processing development of recombinant phenylalanine dehydrogenase

This work presents a comprehensive study between the polymer/salt aqueous two-phase systems (ATPS) and chromatography process for downstream processing of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH). First, the partitioning behavior of recombinant PheDH in polyethylene glycol (PEG)/K₂HPO₄ ATPS was examined. For comparative purpose, a classical chromatographic protocol was performed as well. Investigation of chromatography and ATPS procedures revealed that the ATPS comprising of 9% (w/w) PEG-6000, 16% (w/w) K₂HPO₄ and 16% (w/w) KCl with pH of 8.0, volume ratio (V _R ) of 0.25, temperature of 25 °C and 40% (w/w) cell lysate ensured the most favorable approach for PheDH downstream process. A specific activity of 4,231.4 U/mg, a yield of 96.7% and a recovery of 162.0% were obtained. Furthermore, the shorter process time (4 vs. 48 h) and the lower total cost (4 vs. 20 €) were additionally features that confirmed the suitability of proposed technique.     

Fusarium solani pisi recombinant cutinase partitioning in peg/potassium phosphate aqueous two-phase systems

Summary An aqueous two-phase system of polyethylene glycol (PEG) and potassium phosphate was developed for extraction of a cutinase from cell debris of a recombinant Escherichia coli strain. Basic studies to identify the primary factors which affect cutinase partition, namely the influence of polymer molecular weight, polymer concentration and pH were carried out using a purified preparation of the cutinase. The enzyme partition coefficient was enhanced with decreasing PEG molecular weight, increasing tie-line length and pH.     

Downstream processing of luciferase from fireflies (Photinus pyralis) using aqueous two-phase extraction

The firefly luciferase has been extensively used for sensitive detection of bacteria, gene expression and environmental toxins (biosensors). The aim of the present study was to design a simple and more efficient method for the purification and concentration of luciferase using aqueous two-phase extraction (ATPE). Downstream processing of luciferase from North American Firefly Photinus pyralis was carried out, for the first time, using polymer/salt aqueous two phase system (ATPS) at 4 °C. The enzyme was observed to preferentially partition to the polyethylene glycol (PEG) rich top phase. The best results of purification (13.69 fold) and enzyme activity recovery (118.34%) were observed in the system containing 4.0% (w/w) PEG (1500) and 20.5% (w/w) (NH₄)₂SO₄ with a phase volume ratio of 0.21.     

Response surface methodology for the extraction of wedelolactone from Eclipta alba using aqueous two-phase extraction

Abstract

Aqueous two-phase extraction of wedelolactone from Eclipta alba was studied using the polymer-salt system. The system consisted of polyethylene glycol (PEG) as a top phase (polymer) and sodium citrate as a bottom phase (salt). Process parameters such as PEG concentration, PEG molecular weight, salt concentration, and pH have been optimized using response surface methodology (RSM) with the help of central composite design (CCD). The optimized conditions for aqueous two-phase system (ATPS), in the case of one factor at a time approach, were found as PEG 6000, PEG concentration 18% (w/v), salt concentration 16% (w/v), and pH 7; with maximum extraction yield of 6.52?mg/g. While, RSM studies showed maximum extraction yield of 6.73?mg/g with the optimized parameters as PEG 6000, PEG concentration 18% (w/v), salt concentration 17.96% (w/v), and pH 7. ATPS was found to give a 1.3 fold increase in the extraction yield of wedelolactone as compared to other conventional extraction methods.     

Evaluation of recombinant phenylalanine dehydrogenase behavior in aqueous two-phase partitioning

Recombinant Bacillus sphaericus phenylalanine dehydrogenase (PheDH) partitioning was studied in polyethylene glycol (PEG) and ammonium sulfate aqueous two-phase systems (ATPS). The objectives of this work were to investigate influences; varying the molecular mass and concentration of PEG, pH, phase volume ratio (V_R), tie-line length (TLL) and concentration of (NH₄)₂SO₄ on the partition behavior of PheDH. It was revealed that the partitioning was not affected by V_R, while PEG molecular mass and concentration and (NH₄)₂SO₄ concentration had significant effects on enzyme partitioning. Longer TLL and higher pH resulted in better partitioning into the top phase. Under the most favorable partition conditions with 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH₄)₂SO₄ and V_R = 0.25 at pH 8.0, partition coefficient (K_E), recovery (R%), yield (Y%) and TLL were achieved 58.7%, 135%, 94.42% and 39.89% (w/w), respectively. Overall, the promising results obtained in this research indicated that the ATPS partitioning can be provided an efficient and powerful tool for recovery and purification of recombinant PheDH.     

Potential of Aqueous Two-Phase Systems constructed on flexible devices: Human serum albumin as proof of concept

In the present research, the potential use of flexible disposable devices, specifically blood bags, for the fractionation of biological products using Aqueous Two-Phase Systems (ATPS) polymer–salt is studied and demonstrated. Purified human serum albumin (HSA) was used as model protein. Experiments were carried out on ATPS polyethylene glycol (PEG)–potassium phosphate constructed on rigid recipients (conical tubes) and flexible devices (blood bags). The device used for ATPS construction had no significant effect on HSA partition behavior. Protein partition towards the top phase was favored on systems constructed using PEG 1000 g/mol and TLL 45% (w/w), achieving up to 85% recovery. On the other hand a recovery of 92% was achieved at the bottom phase when PEG 3350 g/mol and TLL 25% (w/w) were used. Human serum was used as a complex sample on ATPS experiments. Selective fractionation of human serum proteins on ATPS constructed on flexible devices was achieved. ATPS constructed on blood bags required short equilibrium times (< 6 min), meaning it is feasible to use this approach on mass scale. The potential use of flexible disposable devices, for the fractionation of biological products using ATPS polymer–salt was demonstrated.