Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems

Thermostable alpha-amylase with temperature optimum at 80 degrees C, molecular mass 58 kDa and pI point 6.9 was purified from a catabolite resistant Bacillus licheniformis strain. The enzyme was sensitive to inhibition by metal ions and N-bromosuccinimide. The partition behaviour of this enzyme in aqueous two-phase systems (ATPS) of the polymer-polymer-water type was investigated and some effects of type, molecular weight and concentration of phase components were studied. Up to 100% retention in the bottom phase of polyethylene glycol 10,000-20,000/dextran 200 system was reached. Best partition conditions were obtained in PEG 10,000-20,000/polyvinyl alcohol 200 systems, where the partition coefficient K increased 750 times to 7.5. Simultaneous production and purification of alpha-amylase and serine proteinase in PEG-polymer-water ATPS were examined. In the system PEG 6,000/ficoll, up to 90% of the amylase was retained in the bottom phase, whereas about 95% of the total protein (K = 22.8) and 60-75% of the proteinase were in the top phase. Similar separation of the enzymes from laboratory supernatant was obtained in system PEG/Na2SO4.     

Production of cyclodextrin homologues using aqueous two-phase system

Cyclodextrin homologues (CDs), produced by cyclodextrin glycosyltransferase (CGTase), were simultaneously partitioned in aqueous two-phase system (ATPS). Partition coefficients of CDs were measured in PEG/salt and PEG/dextran systems. Phosphate, citrate, sulfate were tested as salt. ATPS of PEG/salt and PEG/dextran had the partition coefficients of the CDs, larger than unity. However, PEG/dextran system was observed better than PEG/salt as CGTase activity decreased sharply with salt concentration. Enzymatic reaction occurred mainly in PEG-rich bottom phase because of the low partition coefficient of CGTase. The resulting CDs transferred to the PEG-rich top phase, obeying the diffusional partition. In the ATPS of 7% PEG (M.W. 20,000) and 9% dextran (M.W. 40,000), 7 mg/ml of CDs were obtained in top phase at 4.5 hours.     

Purification and in situ immobilization of lipase from of a mutant of Trichosporon laibacchii using aqueous two-phase systems

The crude intracellular lipase (cell homogenate) from Trichosporon laibacchii was subjected to partial purification by aqueous two-phase system (ATPS) and then in situ immobilization by directly adding diatomites as carrier to the top PEG-rich phase of ATPS. A partition study of lipase in the ATPS formed by polyethylene glycol–potassium phosphate has been performed. The influence of system parameters such as molecular weight of PEG, system phase composition and system pH on the partitioning behaviour of lipase was evaluated. The ATPS consisting of PEG 4000 (12%) and potassium phosphate (K₂HPO₄, 13%) resulted in partition of lipase to the PEG-rich phase with partition coefficient 7.61, activity recovery 80.4%, and purification factor of 5.84 at pH of 7.0 and 2.0% NaCl. Moreover, the in situ immobilization of lipase in PEG phase resulted in a highest immobilized lipase activity of 1114.6 U g^?1. The above results show that this novel lipase immobilization procedure which couples ATPS extract and enzyme immobilization is cost-effective as well as time-saving. It could be potentially useful technique for the purification and immobilization of lipase.     

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.     

Extractive fermentation for enhanced gellan-hydrolysing enzyme production by Bacillus thuringiensis H14

A new extractive fermentation process using PEG and potassium phosphate aqueous two-phase system (ATPS) was developed for enhanced production of gellan-hydrolysing enzyme by Bacillus thuringiensis H14. Five different Bacillus sp. were tested for their ability to synthesize gellan-hydrolysing enzyme. Bacillus thuringiensis H14 was found to be the best organism for gellan-hydrolysing enzyme production. The enzyme showed maximum activity at pH 7.5 and 40 °C. The partition studies of gellan-hydrolysing enzyme in the system using PEG X (X = 9000, 6000, 4000) and potassium phosphate–water and PEG–sodium citrate–water system indicated at PEG (4000)– potassium phosphate–water is the best system for partitioning of gellan-hydrolysing enzyme into the PEG phase (K = 4.99). Gellan-hydrolysing enzyme production by Bacillus thuringiensis H14 was studied in ATPSs composed of PEG X (X = 9000, 6000, 4000) and potassium phosphate. The top phase is continuous and rich in PEG while the bottom phase is dispersed and is rich in phosphate, microbial cells being mainly retained in the bottom phase. The gellan-hydrolysing enzyme produced during fermentation partitioned into the upper PEG phase and total gellan-hydrolysing enzyme produced was 2.12, 2.29 and 2.40 times higher than that of homogeneous fermentation when the fermentations were carried out using PEG 9000–potassium phosphate–water, PEG 6000–potassium phosphate–water, PEG 4000–potassium phosphate–water systems respectively.     

Partitioning of recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) from plant cell suspension culture in PEG/sodium phosphate aqueous two-phase systems

Partitioning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) was achieved in the aqueous two-phase systems (ATPSs) using a crude extract of transgenic tobacco cell suspension culture. This study examined the effects of polyethylene glycol (PEG) molecular weight and concentration and the effects of sodium phosphate concentration in different PEG/sodium phosphate systems on the partition coefficient,K. The best ATPS system was 5% PEG 8,000/1.6 M sodium phosphate after 2 h of incubation at room temperature. In this system, hGM-CSF was partitioned in the PEG-rich phase with a yield of 57.99% andK _hGM-CSF of 8.12. In another system, 3% PEG 10,000/1.6 M sodium phosphate, hGM-CSF was also partitioned primarily in the top phase with a yield of 45.66% andK _hGM-CSF of 7.64 after 2 h of incubation at room temperature.     

Partial purification of α-amylase from culture supernatant of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in aqueous two-phase systems

A study was made of the partition and purification of -amylase from a culture supernatant of Bacillus subtilis in the polyethylene glycol (PEG)—citrate aqueous two-phase system (ATPS). Factors that influenced the partition of the protein in this system, including the molecular weight of the PEG, the tie line length of ATPS, the pH value and the sodium chloride concentration, were investigated. Purification of -amylase was attained with a purification factor (PF) of 1.8 and 90% yield at pH 6.0 in a PEG1000-citrate ATPS with short tie line length. By utilizing the salt-out effect of neutral salt, the purification of -amylase was further improved to 2.0 of PF and 80% yield in a PEG3350-citrate ATPS with 4% sodium chloride.     

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.     

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.     

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.     

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.     

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%.     

Partition behavior of CD133+ stem cells from human umbilical cord blood in aqueous two‐phase systems: In route to establish novel stem cell primary recovery strategies

Aqueous two‐phase systems (ATPS) represent a promising strategy for the recovery of CD133⁺ stem cells. This particular type of stem cells has great potential for research and clinical applications. Traditional [polyethylene glycol (PEG), dextran (DEX), and ficoll] and novel (Ucon) polymer–polymer ATPS were exploited to study the partitioning behavior of CD133⁺ stem cells and contaminants from human umbilical cord blood (HUCB). The aim of the study was to select conditions under which the product of interest and the contaminants concentrate in opposite phases. To accomplish this, three independent samples were tested: (1) enriched CD133⁺ sample, (2) whole HUCB (contaminants), and (3) complex sample (CD133⁺ stem cells and contaminants). The objective of this research was to evaluate the partition behavior of CD133⁺ in ATPS in route to establish the basis for the development of a novel and scalable purification bioprocess. In conclusion, the partitioning behavior of CD133⁺ stem cells and contaminants from complex samples was as follows: 59% of CD133⁺ stem cells fractionated to the top phase when employing ficoll 400,000–DEX 70,000 or 100% to the bottom phase with Ucon‐DEX 75,000 and PEG 8,000‐DEX 500,000 ATPS. In average, 35% of the contaminants partitioned to the top phase of the ficoll 400,000‐DEX 70,000 ATPS, 99% to the dextran rich phase of the Ucon‐DEX 75,000 systems and 97% to the bottom phase of the PEG 8,000‐DEX 500,000. Cell viability was at least 98% after ATPS recovery. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:700–707, 2014     

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.     

Potential application of aqueous two‐phase systems and three‐phase partitioning for the recovery of superoxide dismutase from a clarified homogenate of Kluyveromyces marxianus

Superoxide dismutase (SOD; EC 1.15.1.1) is an antioxidant enzyme that represents the primary cellular defense against superoxide radicals and has interesting applications in the medical and cosmetic industries. In the present work, the partition behavior of SOD in aqueous two‐phase systems (ATPS) (using a standard solution and a complex extract from Kluyveromyces marxianus as sample) was characterized on different types of ATPS (polymer–polymer, polymer–salt, alcohol–salt, and ionic liquid (IL)–salt). The systems composed of PEG 3350‐potassium phosphate, 45% TLL, 0.5 M NaCl (315 U/mg, 87% recovery, and 15.1‐fold purification) and t‐butanol‐20% ammonium sulfate (205.8 U/mg, 80% recovery and 9.8‐fold purification), coupled with a subsequent 100 kDa ultrafiltration stage, allowed the design of a prototype process for the recovery and partial purification of the product of interest. The findings reported herein demonstrate the potential of PEG‐salt ATPS for the potential recovery of SOD. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1326–1334, 2014     

Partitioning studies of α-lactalbumin in environmental friendly poly (ethylene glycol)—citrate salt aqueous two phase systems

Aqueous two-phase systems (ATPS) formed by polymer and salt have been utilized to enrich the desired biomolecule into one of the phase with higher yield and purity. The eco-friendly, biodegradable poly ethylene glycol (PEG) and different citrate salts were chosen as ATPS phase components to investigate the partitioning behavior of α-lactalbumin (α-La). System factors and process parameters such as type and concentration of salt, molecular weight and concentration of PEG, pH, temperature and the effect of additives were studied and the results are discussed in detail. PEG 1000–tri-potassium citrate system yields high partition coefficient of 20 with a better yield of 98 % in the top phase. The addition of NaCl as an additive and acidic pH lowers the yield of α-La in the top phase. Influence of phase volume ratio (V _r) on partitioning was studied and found that the partition coefficient remains almost constant along the tie line. High yield was achieved at a V _r of 3.5 at the tie line length of 50.63 (%, w/w).     

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.     

Scale-up of recombinant cutinase recovery by whole broth extraction with PEG-phosphate aqueous two-phase

A whole broth extraction using an aqueous two-phase system (ATPS) composed by 5% (w/w) PEG 3350 and 15% (w/w) phosphate was used for the scale-up extraction and isolation of a recombinant Fusarium solani pisi cutinase, an extracellular mutant enzyme expressed in Saccharomyces cerevisiae, containing a fusion peptide (WP)₄. The experiments were carried out at three different scales (10 ml, 1 l and 30 l). Mixing time and stirrer speed were evaluated at lab scale (1 l) with two different system compositions. Stirrer speed between 400 and 800 rpm and mixing time between 2 and 5 min led to the highest recoveries of cutinase. In all cases, inclusive of pilot scale (30 l), the equilibrium was reached after a few minutes. The performance of ATPS was reproducible within the scale range of 0.010–30 l and provided a standard deviation of the yield lower than 8%, leading to (i) a partition coefficient over 50, (ii) a yield over 95% and (iii) a concentration factor over 5. The fusion of the peptide (WP)₄ to the cutinase protein enabled a 400 increase of the partition coefficient relative to the wild-type strain.     

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.     

The potential application of aqueous two-phase systems for in situ recovery of 6-pentyl-infinity-pyrone produced by Trichoderma harzianum

Commercial production of aroma compounds by de novo microbial biosynthesis has been principally limited by the low productivity so far achieved. Production of 6-pentyl-alpha-pyrone (6PP), a coconut-like aroma compound, by Trichoderma harzianum has been limited by the toxic effect that occurs even at low concentration (<100 ppm). This work evaluated the feasibility of the use of aqueous-two phase systems (ATPS), as in situ extraction systems, in order to overcome the toxic effects of 6PP and to improve culture productivity. The partition behaviour of 6-pentyl-alpha-pyrone and Trichoderma harzianum mycelium in polyethylene glycol (PEG)-salt and PEG-dextran two-phase systems was investigated and it is reported for the first time. The evaluation of system parameters such as PEG molecular mass, concentration of PEG as well as salt, volume ratio (Vr) and dextran molecular mass, was carried out to determine under which conditions the 6PP partitions to the opposite phase that mycelium does. PEG-dextran systems proved to be unsuitable for the in situ recovery of 6PP because either 6PP and biomass partitioned to the same phase or a large extraction phase was required for the process. ATPS extraction comprising Vr = 0.26, PEG 1450 (7.2% w/w) and sulphate (16.6% w/w) provided the best conditions for the maximum accumulation of the biomass into the bottom phase and concentrated the 6PP in the opposite phase (i.e. 86% of biomass and 56% of 6PP of the total amount loaded from the fermentation extract into the ATPS) for ex situ bioseparation. However, this system caused complete inhibition of the growth of the microorganism during the in situ bioseparation, probably as a consequence of the high ionic strength resulting from the salt concentration. Consequently, two ATPS PEG 8000-sulphate (12%/7% and 6%/14%) were evaluated and proved to be more suitable in the potential application for the in situ recovery of 6PP.