AUTHOR: Laila Sørensen, Andrea Hantke and Niels T Eriksen∗
BACKGROUND: The phycobiliprotein C-phycocyanin (C-PC) is used in cosmetics, diagnostics and foods and also as a nutraceutical or biopharmaceutical. It is produced in the cyanobacterium Arthrospira platensis grown phototrophically in open cultures. C-PC may alternatively be produced heterotrophically in the unicellular rhodophyte Galdieria sulphuraria at higher productivities and under improved hygienic standards if it can be puriﬁed as efﬁciently as C-PC from A. platensis.
RESULTS: Ammonium sulfate fractionation, aqueous two-phase extraction, tangential ﬂow ultraﬁltration and anion exchange chromatography were evaluated with respect to the puriﬁcation of C-PC from G. sulphuraria extracts. Galdieria sulphuraria C-PC showed similar properties to those described for cyanobacterial C-PC with respect to separation by all methodologies. The presence of micelles in G. sulphuraria extracts inﬂuenced the different procedures. Only chromatography was able to separate C-PC from a second phycobiliprotein, allophycocyanin.
CONCLUSION: C-PC from heterotrophic G. sulphuraria shows similar properties to cyanobacterial C-PC and can be puriﬁed to the same standards, despite initial C-PC concentrations being low and impurity concentrations high in G. sulphuraria extracts. �c 2013 Society of Chemical Industry
KEYWORDS: ammonium sulfate fractionation; anion exchange chromatography; aqueous two-phase extraction; ultraﬁltration; phycocyanin, purity number
C-phycocyanin (C-PC) is a blue, light-harvesting phycobiliprotein in cyanobacteria and red algae. It is used in cosmetics and diagnostics because of its strong ﬂuorescence and antioxidant properties,1,2 and there is current interest in the use of C-PC as a dye in foods.3 –5 It has also been demonstrated that C-PC has positive effects on impaired physiological conditions such as inﬂammation,6 ischaemia-induced myocardial injury7 and kidney injury,8 among others.2 C-CP from the cyanobacterium Arthrospira platensis is traded as a nutraceutical or health food.
The colour, the ﬂuorescent properties and the antioxidant and therapeutic potentials of C-PC are related to its covalently bound open chain tetrapyrrole chromophore phycocyanobilin.9 Phycocyanobilin can be enzymatically reduced to phycocyanorubin,10 which is structurally similar to bilirubin,11 a natural antioxidant in plasma that protects lipids from oxidation.12 Bilirubin13,14 and phycocyanorubin11,15 also inhibit the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, reduce the generation of reactive oxygen species in the body and thereby affect a multitude of physiological conditions. For example, elevated plasma bilirubin levels have been linked to lower frequencies of certain cancer forms11 and diabetes,16 while inhibition of NADPH oxidase may protect against inﬂuenza infections.17,18 The oxidised form of bilirubin, biliverdin, is currently extracted from ox bile. Since the availability of this resource will be inadequate to supply biliverdin to large population groups,11 C-PC may be used as a therapeutic replacement for biliverdin.11,15,18 It has been suggested that a regular intake of phycocyanobilin or C-PC will provide protection against cancer11 and other diseases.8 C-PC is presently produced in phototrophic cultures of the cyanobacterium A. platensis (syn. Spirulina platensis)grown outdoors in open ponds or raceways.19,20 Phototrophic cultures always suffer from low productivities, and contamination by foreign organisms cannot be avoided in open cultures. Neither productivities2,21 nor hygienic standards22 of A. platensis cultures therefore meet those of other microbial cultures normally used by industry. Heterotrophic production of C-PC is possible in the unicellular rhodophyte Galdieria sulphuraria,23,24 in isolates that have lost the ability to down-regulate their photosynthetic apparatus and remain pigmented under heterotrophic conditions.25,26 Galdieria sulphuraria grows in ordinary bioreactors where it is possible to avoid contamination and maintain cultures axenic. Light limitation is of no concern in heterotrophic cultures, and volumetric C-PC productivities have been more than ten times higher in heterotrophic G. sulphuraria cultures than in phototrophic A.platensis cultures because of much higher biomass productivities.24 There are no reports on toxins or other harmful products associated wth G. sulphuraria,and this alga was recently evaluated as a potential human food source.27 Galdieria sulphuraria is therefore in many aspects superior to A. platensis when it comes to production of large quantities of C-PC.
The purity of C-PC preparations is conveniently reported by a purity number that describes the ratio between absorbances from phycocyanobilin at 620 nm and all proteins in the preparation at 280 nm, A620/A280. C-PC preparations with purity numbers greater than 0.7 were described as food grade by Rito-Palomares et al.28 while purity numbers of 3.9 and above 4.0 have been considered reactive or analytical grade respectively. Precipitation by ammonium sulfate followed by different chromatographic principles has been used to purify cyanobacterial C-PC from cell extracts to food, reactive and analytical grades.29 –36 Also, ultraﬁltration has been used for puriﬁcation of cyanobacterial C-PC,37,38 and some of the highest purity numbers for C-PC have been obtained in procedures involving aqueous two-phase extraction.24,39 –41
Galdieriasulphuraria may only become an alternative production organism if its C-PC can be puriﬁed as efﬁciently as that from A. platensis. The speciﬁc C-PC content in G. sulphuraria is 10–25 mg g−1, while it can be up to 150 mg g−1 in A. platensis.42 Galdieria sulphuraria is also protected by a cellulose-rich cell wall that makes cell disruption difﬁcult. It is therefore a greater challenge to purify C-PC from G. sulphuraria using the same methodologies as used for puriﬁcation of cyanobacterial C-PC. In this paper we have evaluated a number of methodologies for the recovery of pure C-PC from heterotrophic G. sulphuraria.
Experimental strain and culture conditions
Highly pigmented colonies of G. sulphuraria strain 074G23 were isolated on solid medium2,24 and grown heterotrophically in 1 L conical ﬂasks containing 200 mL of deﬁned growth medium23 supplemented with 5 g L−1 glucose as growth-limiting substrate. Cultivations were carried out in the dark at 42 ◦C and pH 2 and shaken at 200 rpm in an orbital shaker. Cultures were harvested after 8 days, approximately 2 days after they reached stationary phase where the speciﬁc C-PC concentration is maximal.24 Concentrations of C-PC, allophycocyanin (APC) and chlorophyll a were estimated spectrophotometrically at 618, 650 and 664 nm respectively as described by Kursar and Alberte.43 Light scattering from cells or micelles in cultures and cell extracts was subtracted by applying a baseline between the apparent absorbance measurements at 530 and 720 nm respectively. At these wavelengths, absorbance from pigments in G. sulphuraria is minimal.23
Extraction of C-PC
Cells were harvested by centrifugation for 10 min at 5000 × g, washed three times in 50 mmol L−1 potassium phosphate buffer (pH 7.2), resuspended in the same buffer and frozen at −80 ◦C. Frozen cells were disrupted for 2 × 4 min at 3500 rpm in a Mikro-Dismembrater S (B. Braun, Germany, Melsungen), cooled to −80 ◦C in between the two disruption cycles, washed out of the homogenising chamber by 50 mmol L−1 potassium phosphate buffer and centrifuged for 90 min at 25 000 × g. The supernatant was named crude cell extract.
Ammonium sulfate fractionation
Cell debris and proteins in crude cell extracts or in partly puriﬁed C-PC solutions were precipitated by ammonium sulfate at concentrations from 0 to 2 mol L−1, corresponding to 0–50% of the solubility of ammonium sulfate at 20 ◦C. After 30 min of incubation under stirring, the precipitate was pelleted by centrifugation for 30 min at 14 000 × g. The pellet was redissolved in 50 mmol L−1 potassium phosphate buffer (pH 7.2).
Aqueous two-phase extraction
D-PC was separated from cell debris and dissolved cell constituents by aqueous two-phase extraction as described by Patil et al.39 First, 11.63% (w/w) potassium phosphate was added to a solution containing C-PC and the pH was adjusted to 7.2 by 1 mol L−1 H2SO4 or 1 mol L−1 KOH. Then 12.28% (w/w) polyethylene glycol (PEG4000) was added and the volume was adjusted to 3–10 mL by water, resulting in a tie line length of 18.6% (a detailed review on aqueous two-phase systems, including the calculation of tie line lengths, is provided by Raja et al.44). The mixture was stirred at room temperature for 1 h, the two phases were separated by centrifugation for 3 min at 4000 × g and the volumes of the upper PEG4000-rich phase and the lower potassium phosphate-rich phase were quantiﬁed in order to allow measurements of C-PC contents in the two phases. In some experiments the tie line length44 was increased to 33.5% using potassium phosphate and PEG4000 compositions as described by Patil and Raghavarao.40
Ultraﬁltration using 50 or 100 kDa tangential ﬂow ﬁlters (Pellicon XL Ultraﬁltration Module Biomax, Millipore, Billerica, Massachusetts) was used to separate small molecules from C-PC. Between 4 and 10 mL of solutions containing C-PC were diluted in 500 mL of 50 mmol L−1 potassium phosphate buffer (pH 7.2) and pumped through the ﬁlter at a rate of 45 mL min−1. The retentate was repeatedly passed through the ﬁlter until its volume was reduced to 10–20 mL.
Anion exchange chromatography
Anion exchange liquid chromatography was carried out on a HiLoad 26/10 Q Sepharose High Performance column connected to an ¨Akta Puriﬁer System (Amersham Pharmacia Biotech, Uppsala, Sweden) equipped with a UV–visible detector that recorded the absorbance of the efﬂuent at 280, 618 and 650 nm. The column was initially calibrated in 10 mmol L−1 potassium phosphate buffer (pH 7.2), extracts of G. sulphuraria were loaded onto the column, and proteins were eluted by a gradually increasing NaCl concen-tration from 0 to 0.4 mol L−1 in the same buffer used as eluent.
Anion exchange chromatography was also carried out in microcolumns. Q Sepharose Fast Flow resin (Sigma, St. Louis, Missouri) was packed into 1 mL single-use pipette tips; the columns were mounted vertically and washed in 10 mmol L−1 potassium phosphate buffer (pH 7). C-PC solutions were loaded on top and passed through the columns by gravity until the blue colour of C-PC was observed in the efﬂuent. The columns were again washed in 10 mmol L−1 potassium phosphate buffer (pH 7) before C-PC was eluted from the columns by 10 mmol L−1 potassium phosphate buffer (pH 7) containing 0.25 mol L−1 NaCl.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
The purity of C-PC solutions was evaluated by SDS-PAGE using 150 gL L−1 acrylamide/bisacrylamide (37.5:1 v/v) gels. C-PC solutions were mixed with a loading buffer to obtain a ﬁnal concentration of 50 mmol L−1 Tris–HCl (pH 6.8), 144 mmol L−1 mercaptoethanol, 4 g L−1 SDS, 100 g L−1 glycerol and 1 g L−1 bromphenol blue and boiled for 5 min before being loaded onto the gel. Proteins were separated at 200 V and visualised by staining with Coomassie Brilliant Blue.
RESULTS AND DISCUSSION CRUDE EXTRACTS
Batch cultures of G. sulphuraria grew to biomass concentrations of 2.3–2.4 g L−1. When the cells were harvested, glucose had been depleted and the biomass yield on glucose was 0.46–0.48 g g−1, which is similar to yield coefﬁcients reported before.23,24 The speciﬁc C-PC contents reached 25–30 mg g−1 and were thereby comparable to the highest speciﬁc pigment content of 27 mg g−1 seen previously in heterotrophic cultures of this alga.24 The ratio between the speciﬁc concentrations of C-PC and APC, a second phycobiliprotein in G. sulphuraria, was 5:1, as determined spectrophotometrically at 280 nm after chromatic separation of the two pigments.
C-PC concentrations in crude cell extracts of G. sulphuraria were 25–400 mg L−1 depending on the amount of disrupted biomass and the volume of buffer used to wash material out of the homogenising chamber. The purity numbers of C-PC in the crude extracts were below 0.1, which is low compared with the C-PC purity numbers of 0.6–1.18 found in crude extracts of A. platensis and other cyanobacteria.28 –30,32,37,39 The low purity numbers were partly a result of the comparatively low C-PC content in G. sulphuraria24 and partly a result of the need for mechanical grinding of the G. sulphuraria cells, which are protected by a strong wall. The grinding resulted in the formation of micelles26 that were only partly removed by ultracentrifugation. Chlorophyll a and carotenoids dissolved in these micelles contributed to the absorbance at 280 nm. C-PC purity numbers of crude G. sulphuraria extracts can therefore be compared directly with C-PC purity numbers of cyanobacterial extracts only after these micelles are removed.
Ammonium sulfate fractionation
Micelles created during cell homogenisation were efﬁciently precipitated at lower concentrations of ammonium sulfate than needed to precipitate G. sulphuraria C-PC. Ammonium sulfate at concentrations of 0.8–1.2 mol L−1 (20–30% of the saturation concentration) brought the micelles out of suspension, and this was observed as a reduction in the concentration of chlorophyll a (Fig. 1). Galdieria sulphuraria C-PC precipitated only at ammonium sulfate concentrations above 1.28 mol L−1. Less than 10% of the C-PC was lost from supernatants after precipitation of micelles and other impurities using ammonium sulfate concentrations from 0.08 to 1.2 mol L−1.Justas importantly, more than 90% of the C-PC could be recovered after resuspension of pellets created by precipitation in ammonium sulfate concentrations above 1.6 mol L−1. APC co-precipitated with C-PC. In extracts of A. platensis that also contain C-PC in combination with APC, C-PC precipitates at lower concentrations of ammonium sulfate than APC. These two phycobiliproteins can therefore be separated by ammonium sulfate fractionation of cyanobacterial extracts.30 This seems not to be an option with respect to separation of C-CP and APC in extracts of G. sulphuraria.
Figure1. AmountsofC-PC(□), APC (O) and chlorophyll a (◇) in supernatants of Galdieria sulphuraria extracts after addition of increasing concentrations of ammonium sulfate at 20 ◦C, normalised in relation to amounts of pigments in crude cell extract (chlorophyll a was associated with micelles), and amount of C-PC recovered from redissolved precipitates relative to amount of C-PC in crude cell extract ( ). The concentration of C-PC in the crude cell extract before addition of ammonium sulfate was 240 mg L−1.
Aqueous two-phase extraction
The distribution of G. sulphuraria C-PC was investigated in aqueous PEG4000/potassium phosphate two-phase systems with tie line lengths of 18.6 and 33.5%, as these have previously proven suitable for puriﬁcation of C-PC from A. platensis.39,40 PEG4000 makes C-PC preparations viscous and may complicate downstream puriﬁcation procedures, but some of the highest cyanobacterial C-PC purity numbers reported so far have been obtained by the use of aqueous two-phase extraction.24,39 –41 After phase separation, C-PC was predominantly found in the upper PEG4000-rich phase and virtually excluded from the lower potassium phosphate-rich phase. When aqueous two-phase extraction was applied as a ﬁrst step to separate C-PC from other components in crude extracts,28,39 a third intermediate phase formed containing micelles and most of the hydrophilic pigments, but also considerable fractions of C-PC. Formation of this intermediate phase was reduced when micelles had been removed prior to aqueous two-phase extraction by ammonium sulfate precipitation, but not prevented. Recoveries of C-PC from the upper phase were therefore not higher than 56 ± 5% (n = 10) at a tie line length of 18.6%. C-PC recoveries remained at the same level (52 ± 8%, n =6)whenthetieline length was increased to 33.5%. Purity numbers of C-PC solutions, which had previously been recovered after ammonium sulfate fractionation, increased up to 20 times, and purity numbers in the upper phase reached 1.7 ± 0.2 (n = 16). APC was also present in the upper PEG4000-rich phase and not separated from C-PC by aqueous two-phase extraction.
In some trials, potassium phosphate was reintroduced into the PEG4000-rich upper phase after the bottom and intermediate phases had been removed. Thereby the aqueous two-phase extraction procedure with a tie line length of 18.6% was repeated in the same way as described above, but with the contaminants forming the intermediate phase removed. In these cases, C-PC purity numbers and recoveries in the upper phase increased to 2.7 ± 0.1 and 88 ± 3% (n = 2) respectively. These numbers are similar to the purities and recoveries that are obtained by aqueous two-phase extraction of A. platensis C-PC.28 C-PC from G. sulphuraria and that from A. platensis therefore show similar properties in aqueous two-phase systems. The low purity numbers and poor recoveries of C-PC from G.sulphuraria after primary phase separations were caused by impurities in the G. sulphuraria cell extracts that interfered with the separation of phases.
Ultraﬁltration is a method used to remove PEG4000 following aqueous two-phase extraction and other small molecules from C-PC solutions28 and also to increase the concentration of C-PC in solution. More than 50% of G. sulphuraria C-PC was lost in the ﬁltrate when ﬁltered through a 100 kDa tangential ﬂow ﬁlter and when the volume of the C-PC solutions was reduced 25–50 times. When ﬁltered through a 50 kDa ﬁlter, 79 ± 6% (n =5) of the C-PC was retained in the retentate. A certain loss of C-PC during ﬁltration was expected, since similar fractions of cyanobacterial C-PC are also lost during ﬁltration through 50 kDa ﬁlters.37 Even though C-PC is mainly found as αβ trimers and αβ hexamers with molecular weights of 113 and 225 kDa respectively,45 minor fractions will also be dissolved as 38 kDa αβ monomers46 that may pass through the pores of the ﬁlter. The concentration of C-PC and the properties of the solvent affect the fraction of C-PC that will be dissolved as αβ monomers, but it has previously been estimated that approximately 0.1 µmol L−1 or 4 mg L−1 αβ monomers will be present in solutions of G. sulphuraria C-PC.2 Still, ultraﬁltration can be employed for puriﬁcation of G. sulphuraria C-PC with similar efﬁciency as for puriﬁcation of cyanobacterial C-PC.37 A partial loss of C-CP may, however, be expected during ﬁltration processes no matter whether it is of cyanobacterial origin or extracted from G. sulphuraria.
Anion exchange chromatography
Separation of C-PC from APC will probably not be necessary for the use of C-PC in many dietary or therapeutic applications, since it is mainly phycocyanobilin that appears to be the interesting component11,15 and since APC also contains phycocyanobilin groups. However, anion exchange chromatography was still investigated as a method for the separation of C-PC from crude extracts as well as a method for the separation of C-PC from APC. Figure 2A shows the elution of proteins from a crude G. sulphuraria extract loaded onto the column. Proteins eluted from the chromatographic column were recorded at 280 nm, while C-PC was speciﬁcally recorded at its absorption maximum of 618 nm. C-PC eluted at 150 mL at an NaCl concentration of approximately 0.17 mol L−1. The chromatogram recorded at 280 nm indicated that other impurities were still present in the C-PC-containing fractions. Anion exchange chromatography on crude extracts resulted in C-PC solutions with purity numbers of the order of 0.5. The minor peaks observed at 170 mL and 0.22 mmol L−1 NaCl in both chromatograms reﬂect the elution of APC.
When anion exchange chromatography was employed on samples where most other proteins had already been removed by other puriﬁcation procedures, highly pure C-PC virtually free of contaminating proteins, including APC, could be obtained. Figure 2B shows the elution of proteins from a column loaded with a C-PC solution initially puriﬁed by ammonium sulfate fractionation and aqueous two-phase extraction. In this example also the elution of APC was followed at its absorption maximum of 650 nm. Since the absorption spectra of C-PC and APC are not completely separated, elution of both proteins can be seen in all three chromatograms in Fig. 2B, but there is baseline separation between elution of C-PC at 140 mL and APC at 155 mL. SDS-PAGE analysis (Fig. 2B, inset) conﬁrmed the high purity of C-PC and APC recovered in these eluates. C-PC and APC are denatured by SDS, and predominantly the 18 and 20 kDa α and β monomers of C-PC and the slightly smaller α and β monomers of APC are visible in the left (140 mL eluate) and right (155 mL eluate) lanes respectively. Figure 2 demonstrates that preparations of C-PC or APC free from the second phycobiliprotein can be obtained also from extracts of G. sulphuraria. Although C-PC is dominant in G. sulphuraria, pure APC is actually used in a wider range of applications as a ﬂuorescent marker,1 because APC forms the most stable and highly ﬂuorescent αβ hexamers.
Figure 2. Chromatograms of eluted proteins measured at 280 nm (·····), at C-PC absorption maximum of 618 nm (—) and at APC absorption maximum of 650 nm (−−) and NaCl concentration in eluent (—·—). The ﬂow rate of the eluent was 3 mL min−1. (A) Column loaded with crude Galdieria sulphuraria extract. (B) Column loaded with C-PC solution previously puriﬁed by ammonium sulfate fractionation and aqueous two-phase extraction. Inset: SDS-PAGE analysis of fraction eluted at 140 mL, showing 18 kDa α and 20 kDa β monomers of C-PC (left lane), and fraction eluted at 155 mL, showing 17–18 kDa α and β monomers of APC (right lane).
Puriﬁcation of C-PC from heterotrophic Galdieria sulphuraria
The properties of C-PC from G. sulphuraria seem similar to those of cyanobacterial C-PC in relation to the different puriﬁcation methodologies evaluated in this study. It is therefore possible to use and combine the same operational steps and procedures that are used for puriﬁcation of cyanobacterial C-CP also for puriﬁcation of C-PC from G. sulphuraria. The highest purity numbers reported for A. platensis C-PC are between 4.0 and 6.7.29 –31,33,35,36,39 –41 The aim of this study has been to evaluate individual methodologies with respect to puriﬁcation of C-CP from heterotrophic G. sulphuraria in comparison with puriﬁcation of C-PC from phototrophic cyanobacteria. How these methodologies are best combined and optimised for puriﬁcation of G. sulphuraria C-PC will depend on the application and the scale of production. Still, results from exemplary puriﬁcation procedures combining the different methodologies are shown in Table 1. Ammonium sulfate fractionation of crude extracts resulted in C-PC purity numbers of approximately 0.7, which could be increased to 1.1 if the procedure was repeated. These purity numbers are no higher than C-PC purity numbers in crude extracts of A. platensis and other cyanobacteria.28 –30,32,37,39
When ammonium sulfate fractionation was combined with other methodologies, i.e. aqueous two-phase extraction, tangential ﬂow ﬁltration and/or anion exchange chromatography, G. sulphuraria C-PC could be recovered with purity numbers of 3.5–4.5 and recoveries of up to 40% of the amount of C-PC originally present in the crude extracts (Table 1). Ammonium sulfate fractionation combined with anion exchange chromatography, either as initial or ﬁnal procedure, increased purity numbers to 3.5 (the seemingly low yields in experiments involving anion exchange chromatography performed in microcolumns are predominantly the result of the loading procedure, where C-PC was loaded onto the column until a visible amount of C-PC had bled through the column). When ammonium sulfate fractionation was combined with two additional methodologies, C-CP from G. sulphuraria could be puriﬁed to analytical grade with purity numbers above 4.0.
Galdieria sulphuraria offers by far the most productive cultures with regard to the production of C-PC and also the best options for employing high hygienic standards during cultivation.2,24 In this paper we show that the methodologies used for puriﬁcation of cyanobacterial C-PC work equally efﬁciently on C-PC from G. sulphuraria and that G. sulphuraria C-PC can reach purities comparable to those obtained for cyanobacterial C-PC. This is a fundamental prerequisite for future development of large-scale production of this valuable pigment under heterotrophic conditions for use as a food ingredient, dietary supplement, nutraceutical or biopharmaceutical.
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