B-phycoerythrin Research Articles

Single-step extraction/pre-formulation process for B-phycoerythrin using glycerol-based eutectic solvents: A step toward more sustainable production of phycobiliproteins

B-Phycoerythrin (B-PE) is a protein pigment of significant interest to the food and cosmetic industries. B-PE is obtained from algal or microalgal biomass, such as Porphyridium cruentum, after complex pre-treatment, extraction, and pre-formulation schemes. This work investigated the use of a single step process for the extraction and pre-formulation of B-PE from P. cruentum without complex biomass pretreatment. Five hydrophilic NaDES based on glycerol with varying water contents (20–40%) were implemented by ultrasound-assisted extraction using a one-step extraction procedure. The yields obtained were satisfaying, ranging from 5 to 15 mg of B-PE per gram of dry matter. Additionally, a purity index suitable for use in cosmetics and food products was achieved, with an average of >1.5. Furthermore, the extracts obtained in a eutectic medium demonstrated enhanced stability for B-PE, with an average loss of 10% after accelerated aging, allowing storage at room temperature without loss of quality. In particular, NaDES based on glycerol and glucose and/or betaine appeared to be the most effective in preserving the integrity and antioxidant activity of B-PE, with a range of 15–30% quenched DPPH. The ComplexGAPI tool demonstrated that our single-step NaDES process is more environmentally friendly than conventional extraction processes, thanks to lower energy consumption, reduced unitary operations, and less waste production. These results provide a solid foundation for sustainable management of the valorization of P-BE for cosmetic or food industries.

B-phycoerythrin of Porphyridium cruentum UTEX 161: A multifunctional active molecule for the development of biodegradable films

In this study, films were developed combining gelatin-sodium alginate with an aqueous extract of B-phycoerythrin (B-PE) derived from Porphyridium cruentum (45, 67.5, and 90 µg/mL). Firstly, the production conditions of B-PE by the red microalgae were optimized using response surface methodology applied to culture medium composition prior to examining its antioxidant activities. The optimized process yielded a maximum amount of B-PE equal to 4.16 ± 0.24 % of dry matter using an F/2 culture medium supplemented with NaCl = 17 g/L; MgCl2·6H2O = 2.6 g/L and KH2PO4 = 0 g/L. B-PE extracts demonstrated in vitro antioxidant properties, as evidenced by its DPPH radical scavenging capacity, ABTS radical inhibition, and substantial reducing power. B-PE incorporation in gelatin-sodium alginate films resulted in a notable increase in the swelling index and water solubility, and a significant decrease in the moisture content. In addition, when added to gelatin-sodium alginate films, B-PE extracts enhanced ΔE* values and have positive influence on the crystallinity of the developed films as revealed by X‑ray diffraction analysis. Regarding microstructure, films incorporating B-PE extracts exhibited a homogenous structure with a slightly rough surface. Furthermore, these films demonstrated complete biodegradability after 10 days of burial in soil. The current investigation suggested that gelatin-sodium alginate films incorporating B-phycoerythrin obtained from Porphyridium cruentum UTEX 161 could be used as a promising colored functional packaging for food products.

A Chitosan-Based Flocculation Method for Efficient Recovery of High-Purity B-Phycoerythrin from a Low Concentration of Phycobilin in Wastewater.

Increasing the yield and purity of B-phycoerythrin (B-PE) can improve the economic state of microalgae industrial processing. One method of cost reduction involves the recovery of remaining B-PE from wastewater. In this study, we developed a chitosan (CS)-based flocculation technique for the efficient recovery of B-PE from a low concentration of phycobilin in wastewater. We investigated the effects of the molecular weight of chitosan, B-PE/CS mass ratio, and solution pH on the flocculation efficiency of CS and the effects of phosphate buffer concentration and pH on the recovery rate of B-PE. The maximum flocculation efficiency of CS, recovery rate, and purity index of B-PE were 97.19% ± 0.59%, 72.07% ± 1.37%, and 3.20 ± 0.025 (drug grade), respectively. The structural stability and activity of B-PE were maintained during the recovery process. Economic evaluation revealed that our CS-based flocculation method is more economical than the ammonium sulfate precipitation method is. Furthermore, the bridging effect and electrostatic interaction play important roles in B-PE/CS complex flocculation process. Hence, our study provides an efficient and economical method to recover high-purity B-PE from a low concentration of phycobilin in wastewater, which promoted the application of B-PE as a natural pigment protein in food and chemical applications.

Freeze-thaw-assisted aqueous two-phase system as a green and low-cost option for analytical grade B-phycoerythrin production from unicellular microalgae Porphyridium purpureum

Microalgae are promising alternative sources for natural phycobiliprotein production since they grow rapidly and can be cultured on a large scale. However, the overall economic viability of analytical grade phycoerythrin production from microalgae is limited by the high cost of the pretreatment required to break down the cell wall, as well as the expensive reagents and equipment used during purification. This study developed a novel extraction and purification process to recovery B-phycoerythrin (B-PE) in a freeze-thaw-assisted aqueous two-phase system (FT-ATPS) from Porphyridium purpureum. The effects of culture strategy, extraction method and purification factors on B-PE production were studied systematically. The results revealed that low light induced B-PE synthesis, whereas hindered the growth. The stepwise light supplement strategy alleviated the growth limitation under low-light conditions and increased the final B-PE production, up to 182.1 ± 4.1 mg/L (increasing by 73.1 %). Compared with the traditional electricity highly-consumed ultrasonication method, the green freeze-thaw method could extract B-PE without damaging the cell structure and save much more power (>98 %), and the extraction rate reached the highest when frozen at −80 °C. The purity and concentration of B-PE in crude extract were proved to have a significant effect on ATPS purification, and the higher the former, the better the latter. A total production cost of 0.045 $/mg (decreasing by 96.2 %) and purity of 5.6 ± 0.2 (A545/A280) for analytical grade B-PE production were achieved by FT-ATPS based on 4 % polyethylene glycol (PEG2,000) and 20 % ammonium sulfate. Relative to the conventional method of phycoerythrin production, the method developed herein has greater safety, lower cost, and more environmental benefits, which can be function appropriately in the expensive purification of analytical grade phycobiliproteins.

Production characteristics of Porphyridium purpureum (Bory) Drew et Ross semi-continuous culture at low irradiance

The red microalga Porphyridium purpureum (Bory de Saint-Vincent, 1797) Drew et Ross, 1965 is of great interest to researchers as a source of various biologically valuable substances, with their content in cells being determined by cultivation conditions. Phycobiliproteins concentration in P. purpureum cells depends directly on nitrogen concentration in the culture medium and cell irradiance. Semi-continuous cultivation allows maintaining these parameters at a level given. The aim of the work was to study P. purpureum culture growth and B-phycoerythrin (B-PE) accumulation and production at low irradiance, with minimal rates of pigment photodestruction. P. purpureum semi-continuous (quasi-continuous) cultivation was carried out at a specific flow rate of 0.1 and 0.2 day−1 and mean surface irradiance of 5 and 25 W·m−2. P. purpureum culture productivity increased by 1.6–17 times both with a rise in surface irradiance 5 to 25 W·m−2 and an increase in the medium specific flow rate 0.1 to 0.2 day−1. Maximum productivity values for the experimental conditions (0.21 g·L−1·day−1) were recorded at 25 W·m−2 and 20 % medium specific flow rate, but those were 1.5–2 times lower than the precalculated ones. In P. purpureum cells, protein and B-PE concentrations decreased both with an increase in surface irradiance (by 15–20 %) and with a rise in a specific flow rate (by 1.5 times) for all the variants. The shifts in protein and B-PE concentration in P. purpureum culture had a unidirectional character as well; those mainly corresponded to the shift in the culture density. P. purpureum B-PE productivity increased by 1.5–1.9 times with a rise in surface irradiance 5 to 25 W·m−2. Maximum B-PE productivity (13 mg·L−1·day−1) was recorded for the variants of the experiment with a surface irradiance of 25 W·m−2 (0.1 and 0.2 day−1). An increase in specific irradiance of P. purpureum cells 7 to 26 W·g−1 resulted in a rise in biomass productivity by 2.6 times; in B-PE productivity, by 1.8 times; and in protein productivity, by 1.7 times. In the experiment, irradiance was the factor determining the production characteristics of P. purpureum culture, and it was confirmed by the data obtained.

Integration Bioprocess of B-Phycoerythrin and Exopolysaccharides Production From Photosynthetic Microalga Porphyridium cruentum

Red microalgaPorphyridium cruentumhas great potential for converting CO2into high-value bioactive compounds, such as B-phycoerythrin (B-PE) and extracellular polysaccharides or exopolysaccharides (EPS). This study aimed to establish the integration bioprocess of B-PE and EPS production fromP. cruentum. First, different kinds of growth medium and CO2concentration were assessed indoor in terms of high biomass and B-PE and EPS contents. As follows,P. cruentumcells were outdoor scale-up cultured in 700 L pressurized tubular reactors for 9 days till the biomass reached 0.85 g/L and then separated from supernatantsviacentrifugation. Three different methods were adopted to extract phycobiliproteins, and the highest PE contents were extracted from cells by repeated freeze-thawing treatment along with the optimization of significant variables, and finally, 7.99 mg/L B-PE (16,500 Da) with a purity index of 0.82 was obtained. Moreover, analysis of physicochemical properties of EPS extracted fromP. cruentumshowed that the sulfate content was 14.85% and the uronic acid content was 9.36%.

Growth of Porphyridium purpureum (Porphyridiales, Rhodophyta) and Production of B-Phycoerythrin under Varying Illumination

The red pigment B-phycoerythrin (B-PE) belongs to the group of phycobiliproteins (PBPs). It is a part of the light-harvesting pigment complex of the red microalga Porphyridium purpureum (Bory) Drew et Ross, and its amount in cells is determined by the level of irradiation and nitrogen supply. B-PE is a valuable natural pigment whose biotechnological potential is used in nutraceuticals, pharmaceuticals, food and cosmetic industries, and in biomedical research and clinical diagnostics. The dynamics of cell number and the content of photosynthetic pigments are quite often assessed in experiments with P. purpureum. The culture’s density is low in the majority of the experiments, although it is known that it can reach 10 g/L of dry matter. The aim of the study was to investigate the features of the accumulation and production of PBPs in a dense culture of P. purpureum under varying illumination. The molecular-genetic analysis confirmed the taxonomic affiliation of P. purpureum culture. Algae were grown in flat plate photobioreactors with an average illumination of 5, 10, and 15 klx. The dynamics of culture density, the content of B-PE, and production characteristics were used as indicators. It was shown that an increase in the microalgae culture density corresponding to the calculated nitrogen concentration in the nutrient medium was obtained only under illumination of 5 klx. Illumination increased to 10, and 15 klx caused a decrease in both the maximum and average productivities of the culture by 2.5–3.5 times. It was experimentally shown that the highest content of B-PE in the cells and culture of P. purpureum (5.5% of dry matter and 74 mg/L, respectively) was observed under illumination of 5 klx. The pattern of the change in B-PE content in P. purpureum culture was determined depending on the specific illumination of microalgae cells: the pigment concentration hyperbolically decreased with increasing specific illumination. Thus, the level of illumination of P. purpureum microalgae cells had a significant effect on the growth characteristics of the culture (growth rate, B-PE synthesis rate and yield): a lower level of surface illumination was preferred for P. purpureum cultivation. The approach proposed in the study allows one to reduce material costs during the cultivation of P. purpureum while maintaining a high growth rate of the culture.

Laser-Induced Fluorescence Emission (L.I.F.E.) as Novel Non-Invasive Tool for In-Situ Measurements of Biomarkers in Cryospheric Habitats.

Global warming affects microbial communities in a variety of ecosystems, especially cryospheric habitats. However, little is known about microbial-mediated carbon fluxes in extreme environments. Hence, the methodology of sample acquisition described in the very few studies available implies two major problems: A) high resolution data require a large number of samples, which is difficult to obtain in remote areas; B) unavoidable sample manipulation such as cutting, sawing, and melting of ice cores that leads to a misunderstanding of in situ conditions. In this study, a prototype device that requires neither sample preparation nor sample destruction is presented. The device can be used for in situ measurements with a high spectral and spatial resolution in terrestrial and ice ecosystems and is based on the Laser-Induced Fluorescence Emission (L.I.F.E.) technique. Photoautotrophic supraglacial communities can be identified by the detection of L.I.F.E. signatures in photopigments. The L.I.F.E. instrument calibration for the porphyrin derivates chlorophylla (chla) (405 nm laser excitation) and B-phycoerythrin (B-PE) (532 nm laser excitation) is demonstrated. For the validation of this methodology, L.I.F.E. data were ratified by a conventional method for chla quantification that involved pigment extraction and subsequent absorption spectroscopy. The prototype applicability in the field was proven in extreme polar environments. Further testing on terrestrial habitats took place during Mars analog simulations in the Moroccan dessert and on an Austrian rock glacier. The L.I.F.E. instrument enables high resolution scans of large areas with acceptable operation logistics and contributes to a better understanding of the ecological potential of supraglacial communities in the context of global change.

Growth and Biochemical Composition of Porphyridium purpureum SCS-02 under Different Nitrogen Concentrations.

Microalgae of the genus Porphyridium show great potential for large-scale commercial cultivation, as they accumulate large quantities of B-phycoerythrin (B-PE), long chain polyunsaturated fatty acids (LC-PUFAs) and exopolysaccharide (EPS). The present study aimed to adjust culture nitrogen concentrations to produce Porphyridium biomass rich in B-PE, LC-PUFAs and EPS. Porphyridium purpureum SCS-02 was cultured in ASW culture medium with low nitrogen supply (LN, 3.5 mM), medium nitrogen supply (MN, 5.9 mM) or high nitrogen supply (HN, 17.6 mM). HN significantly enhanced the accumulation of biomass, intracellular protein, B-PE and eicosapentaenoic acid. LN increased the intracellular carbohydrate and arachidonic acid content, and promoted the secretion of EPS. The total lipids content was almost unaffected by nitrogen concentration. Based on these results, a semi-continuous two-step process was proposed, which included the production of biomass rich in B-PE and LC-PUFAs with sufficient nitrogen, and induced EPS excretion with limited nitrogen and strong light.

Dye-sensitized solar cells based on natural and artificial phycobiliproteins to capture low light underwater

We developed dye-sensitized solar cells (DSSCs) based on natural and artificial phycobiliproteins (PBPs) to capture low light underwater. We assembled DSSCs with seven types of PBPs as sensitizers and studied their photoelectric properties. The results showed that the PBPs could markedly improve the photoelectrical properties of the DSSCs. The sensitization achieved by B-phycoerythrin (B-PE) from Porphyridium cruentum was superior to that of the other PBPs. The short-circuit current density, open circuit voltage, fill factor, and photoelectric conversion efficiency of the DSSC containing B-PE with a mesoporous titanium dioxide film as a photoelectrode were 3.236 mA/cm2, 0.545 V, 0.569, and 1%, respectively. The DSSC based on B-PE displayed its highest photoelectric conversion efficiency between 525 and 570 nm. However, the maximum photoelectric conversion efficiencies of the DSSCs based on C-phycocyanin from Spirulina platensis and artificial PBPs were observed around 690 nm. DSSCs containing B-PE show great potential for use in underwater photovoltaic applications.

Enhanced B-phycoerythrin production by the red microalga Porphyridium marinum: A powerful agent in industrial applications

The production of B-phycoerythrin (B-PE) from the red microalga Porphyridium marinum was optimized before to purify it and subsequently study its antioxidant activities. NaNO3, K2HPO4 and metal traces concentrations of the culture medium, and luminosity parameters were chosen, according to the Plackett-Burman design, as the most influent factors on the B-PE production by P. marinum. The optimization of these factors according to the Box-Behnken plan gave a maximum of B-PE production equal to 40 mg/g dry weight under the following conditions: NaNO3 = 3.4 g/L; K2HPO4 = 0 g/L; light intensity = 70 μmol photons/m2/s and metal solution = 1.5 mL/L. The B-PE also showed an interesting capacity to chelate Fe3+ (IC50 = 13.91 ± 0.21 μg/mL) and a significant reducing power (OD700nm = 0.485 ± 0.011 at 100 μg/mL). The present study reports the antioxidant potential of purified B-PE from P. marinum that could be potentially used as a source of bioactive protein for a wide range of cosmetic and pharmaceutical applications.

One-step chromatographic procedure for purification of B-phycoerythrin from Porphyridium cruentum

B-phycoerythrin (B-PE) was separated and purified from microalga Porphyridium cruentum using one-step chromatographic method. Phycobiliproteins in P. cruentum was extracted by osmotic shock and initially purified by ultrafiltration. Further purification was carried out with a SOURCE 15Q exchange column and analytical grade B-PE was obtained with a purity ratio (A545/A280) of 5.1 and a yield of 68.5%. It showed a double absorption peaks at 545 nm and 565 nm and a shoulder peak at 498 nm, and displayed a fluorescence emission maximum at 580 nm. The analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) showed a bulky band between 18 and 20 kDa which could be assigned to subunits α and β and a low intensity band of 27 kDa assigned to γ subunit. Our protocol provides attractive alternative to consider for the purification procedure to obtain analytical grade B-PE at commercial level.

Scaling-up of a B-phycoerythrin production and purification bioprocess involving aqueous two-phase systems: Practical experiences

One of the most attractive segments in food and cosmetic industries is that of natural pigments. Since some synthetic pigments have been reported to be hazardous for humans, natural pigments obtained through biotechnological processes represent an attractive alternative. Our research group has previously worked on the development of an aqueous two-phase system (ATPS)-based prototype process for the recovery of B-phycoerythrin (BPE), a natural high-value pigment obtained from Porphyridium cruentum. Detailed studies describing the scaling up of ATPS processes from bench scale to pilot plant facilities are not common. In this paper experiences derived from the scale-up of a previously developed process for production and recovery of highly purified (purity defined as the absorbance ratio A545/A280>4) BPE are described, where a scale-up factor of 850× was implemented. Characterization of cell disruption with a pilot-scale bead mill allowed efficient BPE release at 2900rpm, 10% (w/v) sample load, 60% (v/v) bead load and 0.5mm glass beads and 22min of residence time with a yield of 1.35mg BPE/g of wet biomass. BPE was recovered and purified using a strategy comprising isoelectric precipitation, aqueous two-phase fractionation and ultrafiltration. A 54% global BPE recovery yield, with final purity of 4.1, was achieved under optimal process conditions. Considering total costs for raw materials and energy expenditures for one batch, it was determined that the production cost of BPE was of $1.17USD/mg, which is underneath the commercial price of a BPE standard (>$30USD/mg).

High pressure disruption: a two-step treatment for selective extraction of intracellular components from the microalga Porphyridium cruentum

The microalga Porphyridium cruentum is known to produce many components of interest. One of them is B-Phycoerythrin (B-PE), a water-soluble intracellular pigment used as an immunofluorescent probe. Current methods to extract this molecule involve total cell disruption and lead to a mix of all the water-soluble components. Subsequently, the pigment purification is very complex. An alternative approach to extract B-PE selectively and thus simplify the purification procedure has been developed using a high-pressure cell disrupter. Different pressures (from 27 to 270 MPa), extracting mediums (distilled water and original microalgae culture medium), and numbers of passages (1 to 3) have been tested. Proteins are selectively more extracted than B-PE at low pressure in original medium. It is thus possible to remove part of the intracellular proteins in a first step and then recover enriched B-Phycoerythrin fraction at higher pressure in distilled water.

Energy Transfer from Fluorescent Proteins to Metal Nanoparticles

Energy transfer plays a significant role in numerous chemical, physical, and biological processes. While the use of fluorescent proteins in Forster resonance energy transfer (FRET) studies of biomolecules is common, energy transfer between fluorescent proteins and inorganic nanoparticles has not been explored in detail. In this study, energy transfer from fluorescent phycobiliproteins to noble metal nanoparticles was analyzed. Solutions of B-phycoerythrin (B-PE) were mixed with colloidal Au and Ag nanoparticles and were characterized by steady-state and time-resolved fluorescence spectroscopy to determine the magnitude and mechanism of the energy transfer. It was found that the protein fluorescence was quenched after the addition of metal nanoparticles. Electron microscopy and absorption spectroscopy confirmed that B-PE was adsorbed onto the nanoparticles, creating a favorable geometry for quenching. Time-resolved fluorescence spectroscopy showed that B-PE fluorescence lifetimes decreased from 2.2 ns to 0...

Extraction and purification of B-phycoerythrin from the red microalga Rhodosorus marinus

A description is given of the purification of B-phycoerythrin (B-PE) from the red microalga Rhodosorus marinus. Initially, phycobiliproteins were released from the microalgal cells by manual cellular fragmentation and sonication. B-PE was extracted with ammonium sulfate precipitation, and purified by anionic and size exclusion chromatography. Its purity was tested using indexes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Spectroscopic characterization of B-PE was performed by UV-visible spectroscopy, fluorescence spectroscopy, and circular dichroism. Rhodosorus marinus showed three types of phycobiliproteins: phycoerythrin, phycocyanin, and allophycocyanin. The purified B-PE showed a purity ratio(A545/A280) of 4.8, characteristic peaks at 540 and 562 nm with a shoulder at 498 nm, fluorescence emission maximum at 578 nm, and a secondary structure almost stable with pH changes. B-PE was found to be the predominant pigment in R. marinus and this red microalga could be a viable option for the recovery of this chromoprotein.

A chaotic micromixer modulated by constructive vortex agitation

A novel design for vortex modulation of a passive micromixer, named as the circulation–disturbance micromixer (CDM), has been achieved and analyzed experimentally and numerically. The micromixer consists of slanted grooves on the bottom and a zigzag barrier on the top. In this micromixer, the fluid produces a transverse motion perpendicular to the main field, and two modulated and hyperbolic vortices of disparate size are induced. The active-like agitation produced by the constructive interference of these two vortices induces increased flow through the grooves and the mixing efficiency is hence improved significantly. The 3D flow structure in CDM has been analyzed through both numerical simulation (CFD-ACE+) and two methods of visualization—using dyes and using micro laser-induced fluorescence (μ-LIF, B-phycoerythrin (BPE) and Allophycocyanin alpha subunit (ApcA)) with a confocal microscope. Our results contribute to an understanding of the resulting enhanced hyperbolic flow mixing and provide also a superior microfluidic element for a ‘lab on a chip’. Compared with a slanted groove micromixer, the mixing index of the designed CDM-2T increases 132%, whereas CDM-4T and CDM-8T respectively increase 183% and 280% at Reynolds number 10.

Simplified two-stage method to B-phycoerythrin recovery from Porphyridium cruentum

A simplified two-stage method for B-phycoerythrin (BPE) recovery from Porphyridium cruentum was developed. The proposed method involved cell disruption by sonication and primary recovery by aqueous two-phase partition. The evaluation of two different methods of cell disruption and the effect of increasing concentration of cell homogenate from P. cruentum culture upon aqueous two-phase systems (ATPS) performance was carried out to avoid the use of precipitation stages. Cell disruption by sonication proved to be superior over manual maceration since a five time increase in the concentration of B-phycoerythrin release was achieved. An increase in the concentration of crude extract from disrupted P. cruentum cells loaded to the ATPS (from 10 to 40%, w/w) proved to be suitable to increase the product purity and benefited the processing of highly concentrated disrupted extract. Kinetics studies of phase separation performed suggested the use of batch settlers with height/diameter (H/D) ratio less than one to reduce the necessary time for the phases to separate. The proposed ATPS stage comprising of 29% (w/w) polyethylene glycol (PEG) 1000 g/mol, 9% (w/w) potassium phosphate, tie-line length (TLL) of 45% (w/w), volume ratio ( V R) of 4.5, pH 7.0 and 40% (w/w) crude extract loaded in a batch settler with H/D ratio of 0.5 proved to be efficient for the recovery of 90% of B-phycoerythrin at the top PEG-rich phase. The purity of B-phycoerythrin increased up to 4.0 times after the two-stage method. The results reported here demonstrate the potential implementation of a strategy to B-phycoerythrin recovery with a purity of 3.2 (estimated by the absorbance relation of 545–280 nm) from P. cruentum.

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

AbstractA 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 A545 nm/A280 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 (Vr) 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

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

AbstractA 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 (Vr) 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 Vr = 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; lnKBPE = −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