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Expanded Bed Adsorption Chromatography for

Recovery of Phycocyanins from the Microalga
Spirulina Platensis
Article in Chromatographia December 2005
DOI: 10.1365/s10337-005-0702-9

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Expanded Bed Adsorption Chromatography

for Recovery of Phycocyanins
from the Microalga Spirulina Platensis
2006, 63, 5966

R. Bermejo1, M. A. Felipe1, E. M. Talavera2, J. M. Alvarez-Pez2,&

1
2

Department of Physical and Analytical Chemistry, Jaen University, E.P.S. of Linares, Linares 23700, Spain
Department of Physical Chemistry, Granada University, Granada 18071, Spain; E-Mail: jalvarez@ugr.es

Received: 14 October 2005 / Revised: 23 November 2005 / Accepted: 29 November 2005

Online publication: 3 January 2006

Abstract
We carried out the purification of C-phycocyanin and allophycocyanin from Spirulina platensis
taking advantage of the adsorption properties of the expanded beds. Initially, phycobiliproteins
were released from the microalga cells by osmotic shock. Next, phycocyanins were recovered
by applying the centrifuged cell suspension directly to the anion exchanger Streamline-DEAE
using expanded bed columns, equilibrated with 50 mM sodium phosphate buffer, pH 7.0. After
adsorption, washing was carried out in the expanded-bed mode. Having removed unbound
proteins and cellular debris, the bed was allowed to sediment and phycocyanins rich solution
was eluted with a downward flow of 500 mM sodium phosphate buffer, pH 7.0. Finally, we
utilized conventional gel filtration and ion exchange chromatography methods for separation
and purification of C-phycocyanin and allophycocyanin. The purification steps were monitored
using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the purity of recovered
phycocyanins was confirmed by absorption and emission spectroscopy. The main advantage of
this new method is the high yield achieved in the steps of product extraction and adsorption by
expanded bed adsorption, so reducing both processing times and costs.

Keywords
Electrophoresis
Ion exchange chromatography
Expanded-bed adsorption chromatography
Phycocyanins and Phycobiliproteins
Spirulina platensis

Introduction
Phycobiliproteins are proteins with open
chain tetrapyrrole prosthetic groups
called bilins, linked to the specic cysteine residues in the apoproteins. They
form light-harvesting antenna complexes
and act as photosynthetic accessory pigments in microalgae, the usual source of
these compounds [1]. In terms of their
Original
DOI: 10.1365/s10337-005-0702-9
0009-5893/06/01

absorption properties, the phycobiliproteins are divided into three main classes:
phycoerythrins (PEs, kmax
540570 nm),
phycocyanins (PCs, kmax
610620 nm)
and allophycocyanins (APC, kmax
650
655 nm) [2]. Phycobiliproteins are used as
uorescent markers of cells and macromolecules in biomedical research and
highly sensitive uorescence techniques
[35], they have also been shown to have

a therapeutic value [6] and can be used as

natural dyes in foods and cosmetics [79].
The antenna system of S. platensis is
composed of only C-PC and APC. These
phycobiliproteins consist of equimolar
quantities of two dissimilar subunits, a,
and b, linked to one or more phycocyanobilins [10, 11]. The aggregation state of
the protein in solution is very sensitive to
the pH, ionic strength, and the protein
concentration. At neutral pH, and both
moderate ionic strength and protein concentration, the most common aggregation
state is the trimer. This form conserves the
native protein conformation, and, therefore, its spectral properties. C-PC and
APC have been shown to be particularly
useful in ow cytometry and cell sorting,
using either tunable dye lasers or the less
expensive, higher wavelength helium-neon
lasers [1214]. The fact that the excitation
and emission maxima for APC lie in the
red is particularly important due to the
lack of interfering emissions from most
biological materials in this region of the
spectrum. However, the widespread use of
APC has been somewhat limited by the
high cost of this puried phycobiliprotein.
Conventional schemes for phycobiliproteins purication involve two steps:
the sample pretreatment to liberate the
intracellular material, obtaining a crude
extract ready for the second step where
the phycobiliproteins are separated using
conventional one or more chromatographic processes [1520].
In this article, we carried out the purication of C-phycocyanin and allophycocyanin from Spirulina platensis taking

Chromatographia 2006, 63, January (No. 1/2)

2006 Friedr. Vieweg & Sohn/GWV Fachverlage GmbH

59

advantage of the benecial adsorption

properties of the expanded beds that reduce the number of operations for proteins
adsorption, capturing proteins from particle-containing feedstock without prior
removal of particulates, and enabling
clarication of a cell suspension or cell
homogenate [2124]. This permits the
concentration of the desired product in a
single operation, achieving higher product
recovery in a shorter time. In the rst step,
product extraction from the biomass was
optimized. Next, phycocyanins were
recovered by EBA, obtaining a concentrated eluate ready to be loaded into a
Sephadex column where C-PC and APC
were separated by conventional gel ltration. Finally, purication was achieved by
ion exchange chromatography with
DEAE-cellulose. The purication steps
were monitored using SDS-PAGE, and
the purity of recovered phycocyanins was
conrmed by absorption and emission
spectroscopy. The main advantage of this
new method is that of avoiding product
loss, increasing the yield in the steps of
product extraction and adsorption by
EBA reducing both processing times and
costs. Moreover, this new method proposed could be utilizable in large scale
phycocyanins recovery.

Spectroscopic Methods

Experimental

Protein Extraction

Microalgal Biomass and

Chemicals

For cell disruption we have quantied the

protein extraction under dierent conditions: osmotic shock with 1 M acetic
acid-sodium acetate buer, pH 5.0, osmotic shock with 1 M sodium phosphate
buer, pH 7.0, osmotic shock with distilled water, freeze-thawing, ultrasounds
and lysozyme treatment, as described
elsewhere [24]. All buers contained
0.01% sodium azide. The best results
were obtained by using acetate buer, pH
5.0. Thus, frozen cells of Spirulina platensis were resuspended in 1 M acetic
acid-sodium acetate buer, pH 5.0, at 1
(v/w) buer/biomass ratio, and mixed for
20 min at constant speed by a Heidolph
RZR1 variable-speed magnetic stirrer
(Schwabach, Germany). The resulting
slurry was transferred to centrifuge tubes
and centrifuged at 2500 g for 10 min. The
procedure was repeated twice with the
pellets. The supernatants from three
centrifugations were pooled and dialyzed
against 50 mM sodium phosphate buer,
pH 7.0, obtaining a solution (phycocyanins crude extract) that was stored at

Cells of Spirulina platensis were grown on

the southeast coast of Spain and generously provided by IMADE S.L. (Granada, Spain). For phycobiliproteins
purication, cyanobacterial cells were
removed from the growth medium by
centrifugation and stored at )20 C.
Thus, frozen microalgae cells were used
as the starting material. StreamlineDEAE anion-exchanger, Sephadex G-100
and materials used for SDS-PAGE were
from Pharmacia Biotech (Uppsala, Sweden). Preswollen microgranular DEAEcellulose DE-52 was from Whatman
(Maidstone, UK). Molecular mass standards, dialysis tubing and tubing closures, ammonium sulfate, sodium azide
and all other chemicals were from Sigma
Diagnostics (St. Louis, MO, USA) and
used without further purication. For the
determination
of
the
equilibrium
adsorption isotherm, previously obtained
pure C-PC was utilized [17].

60

4 C and utilized for EBA chromatography experiments after suitable dilution.

Absorbance measurements and absorption spectra were recorded on a

Perkin-Elmer Lambda-20 UV-Vis spectrophotometer (Beaconseld, UK). Fluorescence emission spectra were recorded
on a Jasco FP-6500 spectrouorometer
(Kyoto, Japan). For steady-state polarized uorescence measurements, the
spectrouorometer was equipped with
polarizers in the excitation and emission
paths. Protein concentrations were chosen so that re-absorption of the emission
was negligible. All spectra were recorded
at room temperature. The amounts of CPC and APC in the dierent extracts and
phycobiliproteins-containing
solutions
were calculated using the following
equations to correct the pigments spectral
overlap [25].
C-PCmg mL1 A615 nm  A730 nm
 0:47 A652 nm
 A730 nm =5:34
1
APCmg mL1 A652 nm  A730 nm
 0:208 A615 nm  A730 nm =5:09
2

Phycocyanins Purification
Previous tests using Streamline-DEAE
showed that most contaminant proteins
eluted at 50 mM sodium phosphate buffer, pH 7.0, whereas phycocyanins eluted
at 500 mM sodium phosphate buer, pH
7.0. Consequently, we used 50 mM sodium phosphate buer, pH 7.0, solution
(starting buer) on the adsorption operations.
We determined the adsorption
parameters on the Streamline-DEAE
chromatographic matrix using either pure
C-PC obtained previously [17] or phycocyanins crude extract. We used 15 mL
sealed shake tubes containing either pure
C-PC (assay range between 0 and
9.00 mg C-PC/mL adsorbent) or dilutions of the crude extract (assay range
between 0 and 0.75 mg C-PC/mL adsorbent), in 5 mL of starting buer. The
tubes were placed in a shaking water bath
at 26 C and 1 mL of a 1:1 slurry of
Streamline-DEAE/buer was added to
each tube. After 24 h of equilibration
time the contents of the tubes were analyzed. The equilibrium concentration of
C-PC in the bulk liquid phase was
determined spectroscopically [25] and the
C-PC bound per mL of the ion-exchanger
adsorbent at equilibrium was calculated
by mass balance.
Two dierent columns were used for
EBA experiments: a 2.5 50 cm column
(containing 74 mL of Streamline-DEAE,
giving 15 cm of settled bed height) and a
1.5 50 cm column (containing 27 mL of
Streamline-DEAE giving 15 cm of settled
bed height). Both were equilibrated with
a suitable volume of starting buer. A
Heidolph PD-5001 peristaltic pump
(Schwabach, Germany) regulated the
ow-rate. For the EBA experiments with
the 2.5 50 column, the same 190 mL
volume of sample with dierent protein/
adsorbent ratios (0.81.7 mg C-PC/mL
adsorbent) was used, this being obtained
by dilution of crude extract with a suitable volume of starting buer. The
expansion degree (H/H0) was maintained
constant as far as the double value of its
settled height (H/H0=2). In a typical
experiment, 190 mL of sample was
pumped up the Streamline-DEAE column at constant ow-rate (203 cm h)1)
and washed with the starting buer until

Chromatographia 2006, 63, January (No. 1/2)

Original

the absorbance at 615 nm in the euent

returned to the base line. For the experiments with the 1.5 50 columns the same
100 mL volume of sample with a protein/
adsorbent ratio of 1.3 mg C-PC/mL
adsorbent was used. In this case, dierent
degrees of bed expansion were assayed
(H/H0=1.82.8) by pumping up 100 mL
of sample into the Streamline-DEAE
column at dierent ow-rates (200
407 cm h)1). The column was then washed with the starting buer until the
absorbance at 615 nm in the euent returned to the base line.
After washing in the expanded mode,
the upward ow was stopped and the bed
allowed settling in both columns. The
bound phycocyanins were recovered
using an isocratic elution of 500 mM
sodium phosphate buer, pH 7.0 (86 cm
h)1 downward ow). Fractions of 16 mL
were collected during elution using a
Redifrac fraction collector from Pharmacia Biotech. Phycobiliproteins concentration was determined at 615 and
652 nm and the fractions with a blue
color (phycocyanins rich solution) were
pooled. Next, cleaning-in-place procedure was carried out, the column being
washed with three bed volumes of 0.5 M
NaOH, 1 M NaCl, solution, three bed
volumes of distilled water, three bed
volumes of 25% (v/v) acetic acid, and
three bed volumes of distilled water. The
column was prepared for a new experiment after equilibration with ve bed
volumes of starting buer.
We used the phycocyanins rich solution from the Streamline-DEAE elution
to obtain pure C-PC and APC by means
of the following two-step chromatographic method described previously by
us [17]. The phycocyanins rich solution
from EBA step was dialyzed against a
suitable volume of 50 mM sodium phosphate buer, pH 7.0. The dialyzed solution was applied to a column (2.5
60 cm) of Sephadex G-100, preequilibrated with the same buer, eluted at a
ow-rate of 50 mL h)1 until the eluate
became blue, and collected in fractions of
3 mL. The blue fractions were analyzed
by SDS-PAGE, uv-visible absorption
spectroscopy, and steady-state uorescence. The rst fractions were C-PC rich,
then some fractions in which the two
biliproteins appear clearly mixed and the
latest fractions were APC-rich solutions.
The C-PC- and APC-rich tubes were
pooled separately and brought to 70% of
saturation with (NH4)2SO4, and allowed
Original

to stand for 1 hr prior to centrifugation at

20000 g for 15 minutes. The pellets were
resuspended in starting buer, and then
dialyzed overnight against a suitable volume of the same buer. Each dialyzed
solution was applied to a column (2.5
15 cm) of DEAE-cellulose DE-52 preequilibrated with starting buer. After
washing with 1 bed volume of this buer,
both columns were developed with
290 mM sodium phosphate buer, pH 7.0.
C-PC was eluted from the column loaded
with C-PC-rich solution. On the other
column, the 290 mM eluate was rejected,
and immediately after APC was eluted
with 400 mM sodium phosphate buer,
pH 7.0. The ow-rate was 70 mL h)1. Peak
blue tubes from each biliprotein were
pooled, brought to 70% of saturation with
(NH4)2SO4 and left to stand overnight in
the dark at 4 C before centrifugation. The
pellets of puried biliproteins were resuspended in small volumes of 5 mM sodium
phosphate buer, dialyzed overnight at
4 C against the same buer, and freezedried for storage until utilization.

by osmotic shock by mixing 1 M acetic

acid-sodium acetate buer, pH 5.0, with
biomass paste (18% dry biomass) for
50 min. This operation was repeated
three times for the exhaustive recovery of
the phycocyanins from the biomass paste,
since no absorption was observed from
phycocyanins in the supernatant from a
fourth treatment of the sample. Hence,
the phycocyanins yield in the recovery
step from the intracellular medium can be
estimated around 100%. The yield from
the reference method with lysozyme was
slightly smaller than that from osmotic
shock. The cell homogenate from osmotic
shock was centrifuged to produce the
supernatant (phycocyanins crude extract)
utilized in EBA chromatography. The
absorption spectrum of this extract
(Fig. 1) showed maxima corresponding
to a mixture of phycocyanins (C-PC and
APC) and other contaminant proteins. It
should be emphasized this pretreatment
method is much simpler than others that
use ultrasounds, mechanical breakage or
the adition of chemical compounds
(lysozyme, rivanol, acetone, Triton X100,..) [1520].

SDS-PAGE
Electrophoresis was carried out in a BioRad Miniprotean III vertical slab gel
apparatus (Milan, Italy) using Laemmlibuer system [26] consisting of a 12.5%
(w/v) polyacrylamide slab gel, 0.75 mm
thick, containing 0.1% (w/v) SDS with a
stacking gel of 4% polyacrylamide. Samples were preincubated with 2% (w/v)
SDS, 10% (v/v) glycerol, 4.5% (v/v)
b-mercaptoethanol, 0.025% (w/v) bromophenol blue, and 60 mM Tris-HCl buffer, pH 6.8, for 5 min at 95C. Gels were
run at room temperature and visualized by
staining with 0.1% (w/v) Coomassie Brilliant Blue R-250, 40% methanol (v/v) with
7% (v/v) acetic acid for 30 min, and destained in dilute acetic acid. The following
proteins were used as molecular mass
markers (in kDa): phosphorylase b (94.0),
albumin (67.0), ovalbumin (43.0), carbonic anhydrase (30.0), trypsin inhibitor
(20.0) and a-lactalbumin (14.4).

Results and Discussion

Extraction
The inuence of cell disruption methods
on the recovery of phycobiliproteins was
studied and the best results were obtained
Chromatographia 2006, 63, January (No. 1/2)

Expanded Bed
Chromatography
We determined the equilibrium adsorption isotherms on the Streamline-DEAE
matrix by plotting the C-PC adsorbed per
volume of Streamline matrix vs the equilibrium concentration of phycobiliprotein
in buer. Fig. 2 shows the plot of the
isotherms using either pure C-PC obtained previously [17] or phycocyanins
crude extract. The agreement with
Langmuirs model was very good. From
the non linear least squares tting of the
model to experimental data of Fig. 2
we recovered the Langmuir parameters
(r2 = 0.99 in both cases). The maximum
equilibrium binding capacity was 11.7
and 0.8 mg C-PC/mL adsorbent for pure
C-PC and crude extract, respectively.
These data indicate that the capacity of
the adsorbent decreases considerably in
the presence of other contaminant proteins in the crude extract. Unfortunately,
ligands with the requisite high specicity
for the target protein often cannot withstand the rigorous cleaning-in-place procedures that are frequently required when
processing crude feedstock.
We utilized two expanded bed chromatography columns with dierent

61

0.7
0.6

Absorbance

0.5
0.4
0.3
0.2
0.1
0.0
300

400
500
Wavelength (nm)

600

700

Fig. 1. (- - -) Absorption spectrum of phycocyanins crude extract and () phycocyanins rich

solution from expanded bed adsorption chromatography

Adsorbed C-PC (mg mL-1)

0
0

10

20

30

40

50

60

70

[C-PC] (mg L-1)

0.7

B
Adsorbed C-PC (mg mL-1)

0.6
0.5
0.4
0.3
0.2
0.1
0.0
0

10

20

30

40

50

60

[C-PC] (mg L-1)

Fig. 2. Equilibrium adsorption isotherms on the Streamline-DEAE matrix, using pure C-PC (A)
and phycocyanins crude extract (B)

internal diameters (I.D. 2.5 and 1.5 cm).

The relative increase of the gel height vs
ow-rate was linear in both columns
(correlation coecients were 0.99) in the
assayed range (150410 cm h)1).

62

EBA Chromatography at Constant H/H0

using a 2.5 cm I.D. Column

Application of the crude extracts without

dilution to the EBA systems usually causes
ow disturbance and unequal distribution

of the sample, due to its high viscosity [21].

Thus, the inuence of the viscosity of the
sample on system behavior, with reference
to the yield of the EBA step, was studied by
testing dierent protein loadings at different crude extract concentrations. In
all experiments 190 mL of phycocyanins
crude extract solution was applied to the
column. We used a range between 0.8 to
1.7 for the C-PC (mg)/adsorbent (mL) ratios. This range is higher than the adsorption capacity measured for C-PC in
Streamline-DEAE, since the adsorption
capacity is usually higher in dynamic mode
than in static [27].
Another signicant parameter is the
expanded bed height. In these experiments an expanded bed with double its
settled height (obtained by a ow-rate of
203 cm h)1) was used, since this ow-rate
provided good results in a similar system
[24]. Moreover, at the best sample viscosity, the behavior of the expanded bed
column, at the above mentioned owrate, was more similar to plug ow and
no channeling phenomena were observed.
This ow-rate decreased both the mass
transfer resistance of the puried bioproduct at the adsorbent surface and the
processing time, thus improving system
productivity [21, 24].
The data in Table 1 were obtained under these operational conditions. These
data show that the yield of the process had
a maximum value at 1.3 C-PC/adsorbent
ratio, resulting in 80 % and 95 % for C-PC
and APC recovery, respectively. Thus,
when the concentration of the sample was
up or below this maximum value, the yield
decreased for both phycobiliproteins. It is
clear that the decrease in diusion rate
caused by the increase in viscosity aects
the adsorption of proteins to the gel particles, whereas the excessive sample dilution allows protein loss resulting in a
decrease in the adsorption yield. The
remaining unbound molecules were removed by washing the bed in the expanded
mode using a twelve-fold bed volume of
starting buer. In this operation, the
supercial velocity was changed smoothly
in order to maintain a constant degree of
bed expansion.
The adsorbed phycocyanins were
eluted using 500 mM sodium phosphate
buer, pH 7.0. Elution from StreamlineDEAE was assayed in expanded bed and
packed bed modes. In both cases 190 mL
of sample were applied to an expanded
bed volume of 150 mL under similar
conditions to the adsorption and wash-

Chromatographia 2006, 63, January (No. 1/2)

Original

EBA Chromatography at Variable

H/H0 using a 1.5 cm I.D. Column

As has been pointed out in recent research, the scale down could improve the
yield recovery [27]. In order to test the
possibility of scaling down the process
and to nd better column geometry for
phycocyanins purication, 1.5 cm I.D
columns were employed. In these experiments, the columns were loaded with
100 mL of phycocyanins crude extract,
maintaining the mg C-PC/ mL adsorbent
Original

8
Sample load and wash

Elution

7
Absorbance (615 nm)

ing. Elution in expanded bed mode needed 640 mL of mobile phase to elute the
protein, while, in packed bed mode, only
160 mL was necessary to recover the
same quantity of protein. Thus, the
sample in packed bed mode was 4 times
more concentrated than in expanded bed
mode. Particulate material was not observed in the eluted fractions. Therefore,
it was not necessary to centrifuge the
samples to obtain their UV-Vis spectra.
The absorbance of the fractions obtained
shows a sharp peak (Fig. 3, Elution),
indicating high elution eciency. Both
the loading sample/expanded bed ratio
(expressed as volume) and the concentration factor (expressed as volume of
expanded bed/volume of sample eluted)
were similar to those reported previously
in the presence of cells [27, 28]. The UVVis absorption spectrum of phycocyanins
rich solution after the packed bed elution
stage corresponds to a biliprotein mixture
constituted for C-PC and APC (Fig. 1).
The spectrum showed a signicant loss of
absorption in the near-UV region with
respect to the phycocyanins crude extract
spectrum. Moreover, the A615/A280 ratio>2, is characteristic of phycocyanins
rich solution and three times higher
than that corresponding to phycocyanins
crude extract (A615/A280 = 0.6).
The best recovery obtained was 80%
and 95% for C-PC and APC respectively,
indicating that losses of phycocyanins
during the ow-through and wash stages
were very low (Table 1). It should be
emphasized that it was more important to
achieve a high phycocyanins recovery than
a high purity product, since the procedure
is intended to replace low yield steps that
result in phycocyanins loss. In the method
described here, one cycle of the concentration process by EBA chromatography,
including equilibration (30 min) + loading (12 min) + washing (120 min) + elution (20 min), took only about 3 h.

6
5
4
3
2
1
0
0

500

1000

1500

2000

2500

Volume (mL)

Fig. 3. Absorbance at 615 nm of euent from the column (50 2.5 cm), during EBA
chromatography of crude extract from S. platensis biomass. 190 mL of phycocyanins crude
extract (1.3 mg CPC/mL adsorbent) was applied to Streamline-DEAE column. Feed rate =
203 cm h)1, elution rate = 86 cm h)1. In the elution step, the absorbance was measured after
suitable eluate dilution. Thus, the absorbance values in ordinate axes are the resulting values after
multiplication by the dilution factor

Table 1. Inuence of the loading and biliprotein/adsorbent ratio on the phycocyanins recovery
percentage upon elution in Streamline-DEAE. In all experiments the sample volume loaded was
190 mL. Column = 50 2.5 cm, H/H0 = 2 and ow-rate = 203 cm h)1
C-PC loaded
(mg)

APC loaded
(mg)

C-PC/adsorbent
ratio (mg mL)1)

C-PC
recovery (%)

APC
recovery (%)

126
97
75
60

105
81
62
50

1.7
1.3
1.0
0.8

68
80
77
73

85
95
93
89

Table 2. Inuence of the dierent degrees of bed expansion on the phycocyanins recovery percentage upon elution in a 50 1.5 cm Streamline-DEAE column. In all experiments the sample
volume loaded was 100 mL and the C-PC/adsorbent ratio was 1.3 mg mL)1.
H/H0

Flow-rate (cm h)1)

C-PC recovery (%)

APC recovery (%)

1.8
2.0
2.2
2.4
2.6
2.8

200
230
272
339
383
407

41.0
69.4
70.5
67.5
64.6
64.0

70.0
77.4
77.8
75.9
75.6
76.0

ratio, at the optimum value of 1.3, previously obtained with the 2.5 cm I.D.
column. In addition, dierent degrees of
bed expansion were assayed. To obtain
the desired degrees of expansion (H/
H0=1.82.8), dierent ow-rates (200
407 cm h)1) were necessary. The data in
Table 2 show that when the expanded
bed volume was between 2 and 2.2 times
the settled bed volume, the best results
were achieved. Yield decreased for both
phycobiliproteins with increasing expansion degree. When expansion degree increases, it is clear that an increase in the
distance between matrix particles occurs,
which causes a minor adsorption of proChromatographia 2006, 63, January (No. 1/2)

teins to the gel particles. These results

corroborate the ow-rate and expanded
bed height employed in the process with
the 2.5 cm I.D. columns. Moreover,
when compared with Table 1, they also
indicate that scale down was ineective
(yield decrease with decreasing column
diameter).

Conventional Packed Bed

Chromatography
In order to obtain pure C-PC and APC,
the phycocyanins rich solution recovered
from the EBA step was utilized as start-

63

Fig. 4. SDS-PAGE characterization of dierent protein solutions. Lane 4 = molecular mass

markers, lane 3 = phycocyanins crude extract, lane 2 = eluate after EBA chromatography,
lane1 = eluate after packed bed chromatography

ing material. The experimental results

indicate that the best yield is achieved
with the two-step chromatographic
method described by us elsewhere [17]
and overviewed in the Experimental
section. We have also studied the purication through only one step by DEAEcellulose chromatography. Continuous
and discontinuous ionic strength gradients were assayed, as well as pH gradients. Nevertheless, the purication of
both proteins was not achieved at the pH
conditions where the proteins were stable.
Only pure C-PC was recovered using an
ionic strength discontinuous gradient. We
therefore followed our previous methodology and pure C-PC and APC were
recovered after DEAE-cellulose chromatography. The achieved yield was 12%
and 10% in C-PC and APC, respectively,
expressed as the percentage of pure biliprotein in the nal eluate after this third
step, per biliprotein loaded onto the gel
ltration matrix [17].

Purity Test
To monitor the purication steps, each
peak from the elution stages was developed by SDS-PAGE electrophoresis.
Unfortunately, the a- and b-subunits of
C-PC and APC have very similar
molecular
masses
(21.5 kDa
and
19.0 kDa, and 19.6 kDa and 17.7 kDa,
respectively), both being present in the
same amount [17]. Consequently, their
mobility is very similar and it is dicult

64

to separate them with the simple method

employed. Fig. 4 shows that unwanted
proteins were removed at each successive
chromatographic step (lanes 3 to 1). The
bands in lanes 2 and 3 should be
assigned to the combination of C-PC
and APC a-b-subunits (intense bands)
along with other contaminant proteins,
these bands being less intense than biliprotein bands. It can be observed that
many of the unwanted proteins from
crude extract (lane 3) were removed after
EBA chromatography (lane 2). Similarly, the eluate from DEAE-cellulose
(lane 1) yields only one, poorly resolved,
bulky band which should be assigned to
the combination of a-subunit and bsubunit from C-PC, in agreement with
the molecular mass markers.
Phycocyanins purity and their conformational state were tested using
spectroscopic
characterization.
For
comparative spectral analysis the phycocyanins blue fractions were pooled
and treated as described previously [17].
Fig. 5 shows absorption, uorescence
and anisotropy spectra from C-PC and
APC dialyzed solutions. The absorption
spectra of the trimeric and monomeric
C-PC forms are only slightly aected by
the aggregation changes in the protein
[20]. Thus, the absorption and uorescence spectra from the dialyzed C-PC
solutions in Fig. 5 agree well with those
published for pure C-PC [17, 2931],
although it is not possible to recognize
the aggregation state from these spectra.
On the contrary, the dissociation of

trimeric APC into monomeric APC

causes the absorption maximum to shift
from 652 2 nm to 613 2 nm and
the emission maximum to shift from
658 2 nm to around 636 4 nm
[17,30,3235]. The dialyzed APC solutions showed no absorbance and
uorescence peak other than that corresponding to the trimeric APC. Hence,
the APC puried in our experiments
retained its native trimeric structure.
The easiest and most explanatory
spectroscopic test to determine the
aggregation state of biliproteins is to record the excitation uorescent steadystate anisotropy spectrum. Since energy
transfer between chromophores modulates the uorescent properties of these
proteins, a dramatic increase in the
anisotropy spectra has been observed
when the aggregation state of the biliproteins changes from trimers to monomers [17,18,31], due to the chromophores
uncoupling, which altered the pathways
for energy transfer between them. The
spectra shapes and the quantitative values of the C-PC and APC anisotropy in
Fig. 5C are similar to those published
earlier for trimeric C-PC and APC from
S. platensis [17, 36].
It should be emphasized that the yield
of the overall phycocyanins purication
process was 9.6% for C-PC and 9.5% for
APC. This value is higher than the value
obtained previously (around 1%) using
methodologies that not involve EBA
chromatography [17, 35] but it would be
greater if the last packed bed step yield
increases. We are currently conducting
experiments in order to optimize the
separation and purication of C-PC and
APC.

Conclusions
We have achieved C-PC and APC purication from the blue alga S. platensis
by
EBA
chromatography
using
Streamline-DEAE, complemented by
a conventional two-step chromatographic method. Only a simple low cost
pretreatment was necessary to optimize
the expanded bed step. The use of EBA
followed by packed bed elution in the
same column eliminates many steps and
produces concentrated and partially
puried protein ready for use in the
next purication step. The work presented here provides pure C-PC and
APC solutions in trimeric state. The

Chromatographia 2006, 63, January (No. 1/2)

Original

0.6

100

0.5
80
Fluorescence Intensity

Absorbance

0.4

0.3

0.2

60

40

20

0.1

0.0

0
300

350

400

450

500

550

600

650

700

560

580

Wavelength (nm)

600

620

640

660

680

700

Wavelength (nm)

0.12

C
0.10

Anisotropy

0.08

0.06

0.04

0.02

0.00
500

525

550

575

600

625

650

675

Wavelength (nm)
Fig. 5. Spectroscopic characterization of puried C-PC and APC in 5 mM sodium phosphate buer, pH 7.0. (A) Absorption spectra of () C-PC and
( ) APC. (B) Fluorescence emission spectra of () C-PC and ( ) APC. Absorption and uorescence spectra were normalized since these were
only used on a comparative basis. (C) Excitation uorescence anisotropy spectra of (m) C-PC and (d) APC. The anisotropy values are the arithmetical
average of ve anisotropy measurements and the error bars are the standard deviations of these anisotropy readings. All spectra were recorded at room
temperature

purication procedure shown simplies

the product isolation process, reduces
processing time and utilities consumption, and increases total product yield.

Acknowledgements
We thank Imade S.L. for providing us
with Spirulina platensis raw material.

Original

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