Journal of Biotechnology 132 (2007) 75–81
Characterization of a ?-fructofuranosidase from Schwanniomyces
occidentalis with transfructosylating activity yielding
the prebiotic 6-kestose
Miguel´Alvaro-Benitoa, Miguel de Abreua, Luc´ ıa Fern´ andez-Arrojob,
Francisco J. Ploub, Jes´ us Jim´ enez-Barberoc, Antonio Ballesterosb,
Julio Polainad, Mar´ ıa Fern´ andez-Lobatoa,∗
aCentro de Biolog´ ıa Molecular Severo Ochoa, Departamento de Biolog´ ıa Molecular (CSIC-UAM), Universidad Aut´ onoma Madrid,
Cantoblanco, 28049 Madrid, Spain
bDepartamento de Biocat´ alisis, Instituto de Cat´ alisis y Petroleoqu´ ımica, CSIC, Cantoblanco, 28049 Madrid, Spain
cCentro de Investigaciones Biol´ ogicas, CSIC, Ramiro de Maeztu, 28040 Madrid, Spain
dInstituto de Agroqu´ ımica y Tecnolog´ ıa de Alimentos, CSIC, Paterna, 46980 Valencia, Spain
Received 19 December 2006; received in revised form 15 March 2007; accepted 20 July 2007
Schwanniomyces occidentalis. The enzyme shows broad substrate specificity, hydrolyzing sucrose, 1-kestose, nystose and raffinose, with different
catalytic efficiencies (kcat/Km). Although the main reaction catalysed by this enzyme is sucrose hydrolysis, it also produces two fructooligosaccha-
rides (FOS) by transfructosylation. A combination of1H,13C and 2D-NMR techniques shows that the major product is the prebiotic trisaccharide
enzymes, both at the kinetic maximum (76gl−1) and at reaction equilibrium (44gl−1). The total FOS production in the kinetic maximum was
101gl−1, which corresponded to 16.4% (w/w) referred to the total carbohydrates in the reaction mixture.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Schwanniomyces occidentalis; Invertase; ?-Fructofuranosidase; 6-Kestose; Fructosyltransferase; Prebiotic oligosaccharides
Enzymes that hydrolyse sucrose are collectively referred
to as invertases or ?-fructofuranosidases (EC 184.108.40.206), and
they catalyse the release of ?-fructose from non-reducing
termini of various ?-d-fructofuranoside substrates. Yeast ?-
cerevisiae (Taussig and Carlson, 1983; Reddy and Maley, 1990,
1996), Schizosaccharomyces pombe (Moreno et al., 1990),
Pichia anomala (Rodr´ ıguez et al., 1995; P´ erez et al., 1996),
Candida utilis (Ch´ avez et al., 1998) and Arxula adeninivorans
(Boer et al., 2004). In general, these enzymes exhibit a high
∗Corresponding author. Tel.: +34 91 4978052; fax: +34 91 4978087.
E-mail address: firstname.lastname@example.org (M. Fern´ andez-Lobato).
degree of sequence homology, and based on their amino acid
sequences, they fall into family 32 of the glycosyl-hydrolases
(GH) (Coutinho and Henrissat, 1999). They share two sig-
nificant located acidic residues, which are necessary for the
displacement mechanism in which a covalent glycosyl-enzyme
intermediate is formed. Furthermore, the three-dimensional
(var. awamori) (Nagem et al., 2004) have been also reported.
In addition to releasing d-glucose and d-fructose from
sucrose, microbial ?-fructofuranosidases may catalyse the syn-
to three fructosyl moieties are linked to the sucrose by different
0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
M.´Alvaro-Benito et al. / Journal of Biotechnology 132 (2007) 75–81
Polakovic, 2001). FOS containing ?-(2→1)-bonds (1-kestose,
nystose and 1F-fructofuranosylnystose) are produced commer-
cially in vast quantities as their prebiotic properties, and exert
a beneficial effect on human health because they are selectively
fermented by colonic flora (Ghazi et al., 2005).
There is great interest in the development novel FOS with
improved prebiotic and physiological properties. In this con-
text, ?-(2→6)-linked FOS (6-kestose being first in the series)
were metabolized by different Bifidobacteria strains when sup-
plied as the sole carbon source (Marx et al., 2000). The FOS
were synthesized by acid hydrolysis of ?-(2→6)-linked poly-
mers containing a glucose at one terminus (levans), these being
produced by several microorganisms growing in sucrose-based
medium (Bekers et al., 2002). The discovery of novel enzymes
that synthesize ?-(2→6)-linked FOS from sucrose may, how-
ever, provide a non-pollutant alternative to acid hydrolysis of
extracellular enzymes. It has been extensively used in biotech-
nology, where it has a high potential for enzyme production.
Several of its amylolytic enzymes (glucoamylase and amylases)
et al., 1990; Y´ a˜ nez et al., 1998) and invertase, one of the pre-
dominant secreted proteins when lactose is used as the carbon
source (Klein et al., 1989a; Costagliogli et al., 1997), puri-
fied (Klein et al., 1989a). The invertase gene encodes a 533
amino acids protein, which shares similar sequence similarity
with other invertases (Klein et al., 1989b). In this work, we
have studied the biochemical properties of Sw. occidentalis ?-
fructofuranosidase, and kinetic studies of the hydrolase activity
were developed using different substrates. The fructosyltrans-
ferase ability of this enzyme was investigated in detail.
2. Materials and methods
2.1. Materials, organisms and growth conditions
Sucrose and fructose were purchased from Merck. 1-Kestose
and nystose were from TCI Europe (Zwijndrecht, Belgium). All
other reagents and solvents were of the highest available purity
and used as purchased.
Sw. occidentalis (formerly Debaryomyces occidentalis)
strain employed was ATCC26077. Yeasts were grown at 29◦C
on YEPD (1%, w/v, yeast extract, 2%, w/v, peptone, 2%,
w/v, glucose) or Lactose Medium (0.3%, w/v, yeast extract
from Difco, 0.35%, w/v, bactopeptone, 0.5%, w/v, KH2PO4,
0.1%, w/v, MgSO4·7H2O, 0.1%, w/v, (NH4)SO2, 2%, w/v, lac-
tose). Growth was monitored spectrophotometrically at 660nm
2.2. Protein purification and quantification
Invertase (0.4Uml−1) secreted by Sw. occidentalis growing
in 1l culture (Lactose Medium, 60h, A660nm=8.31) was con-
centrated and fractionated by filtration through 30000MWCO
PES Viva Flow 50 system (Vivascience). The active fraction
(15ml) was dialysed against 20mM sodium phosphate, pH 7.0
(buffer A) and applied to DEAE-Sephacel (30ml) equilibrated
with buffer A. The protein was eluted with a 0–0.5M NaCl gra-
dient at a flow rate of 1.25mlmin−1. Active fractions (4.7ml)
eluting at 0.2M NaCl were dialysed against 20mM sodium
acetate, pH 5 (buffer B) and applied to DEAE-Sephacel equili-
brated with buffer B. The protein was eluted as above and active
were pooled and stored at −20◦C. All the procedures were per-
formed at room temperature (22◦C). Protein concentration was
determined with the Bio-Rad microprotein assay, in accordance
with the manufacturer’s specifications, with bovine serum albu-
min as a standard. Coomassie-stained SDS-PAGE (8%) of the
samples confirmed the invertase purification. Invertase activity
was detected from native preparation using electrophoresis on
2,3,5-triphenyltetrazolium chloride in 0.25M NaOH as previ-
(Novozymes) was used as a control.
The native molecular mass of invertase was estimated by
filtration in a Sephadex G-200 (2.5cm×45cm) column equi-
librated and eluted with 100mM Tris–HCl, pH 7.0. Elution
was performed at a flow rate of 1mlmin−1. Fractions of 3ml
were pooled. The void volume of the column was determined
using blue dextran. ?-Amylase (200kDa), alcohol dehydro-
genase (150kDa), bovine serum albumin (67kDa), carbonic
anhydrase (29kDa) and cytochrome C (12.4kDa) were used
for column calibration, with elution volumes of 48, 69, 81, 132
2.3. Enzyme and kinetic analysis
Kinetic constants were determined by measuring the release
of reducing sugars from different substrates by the dinitrosali-
cylic acid (DNS) method adapted to a 96-well microplate scale
(Ghazi et al., 2006). The reaction mixture (50?l) contained the
substrate in 0.2M sodium acetate buffer (pH 5.6). Substrate
concentration was varied in the range 0–16mM for sucrose,
0–10mM for 1-kestose, 0–8mM for nystose and 0–12mM for
raffinose. A calibration curve was performed with a 2gl−1glu-
was incubated at 50◦C and 200rpm for 5–20min in an orbital
shaker (Stuart Scientific). Then, 50?l of 10gl−1DNS were
added to each well. The plate was sealed with a seal plate tape
Then, the microplate was cooled, 150?l of water added to each
reader (model Versamax, Molecular Devices). One unit (U) of
activity was defined as that catalysing the formation of 1?mol
reducing sugar per minute under the above conditions.
Curve plotting and analysis of the curves was carried out
using SigmaPlot program (version 7.101). Kinetic param-
eters were calculated fitting the initial rate values to the
M.´Alvaro-Benito et al. / Journal of Biotechnology 132 (2007) 75–81
Estimation of hydrolase activity at different pH and temper-
ature values was carried out under the above conditions using
(pH 3.5–4.5), Na2HPO4/NaH2PO4(pH 4.5–7.5) and Tris–HCl
(pH 7.5–9.0), all 100mM.
2.4. Batch production of fructooligosaccharides
The Sw. occidentalis ?-fructofuranosidase was added to a
sucrose solution (600gl−1, 1.75M) in 0.2M sodium acetate
buffer (pH 5.6). Total reaction volume was 2ml. The final activ-
standard DNS microassay using 30mM sucrose). The mixture
At different times, 40?l aliquots were extracted, diluted with
160?l water and incubated for 10min at 90◦C to inactivate the
enzyme. Samples were centrifuged for 5min at 3500×g using
and analysed by HPLC.
2.5. HPLC analysis
The concentration of the different products was analysed
by HPLC with a quaternary pump (Delta 600, Waters) cou-
pled to a 5?m Lichrosorb-NH2 column (4.6mm×250mm)
(Merck). Detection was performed using an evaporative light-
scattering detector DDL-31 (Eurosep) equilibrated at 85◦C.
Acetonitrile:water 85:15 (v/v), degassed with helium, was used
this eluent to acetonitrile:water 75:25 (v/v) was performed in
2min, and held for 6min. A new gradient to acetonitrile:water
70:30 (v/v) was performed in 5min and held for 14min. Total
analysis time was 35min. The column temperature was kept
constant at 25◦C. The data obtained were analysed using the
2.6. Purification of 6-kestose
Sucrose (600gl−1, 1.75M) in 0.2M sodium acetate
buffer (pH 5.6) was mixed with pure Sw. occidentalis ?-
fructofuranosidase to a final enzyme concentration of 2Uml−1.
The mixture was incubated at 50◦C with orbital shaking
(200rpm) during 3 days. Then, the reaction mixture was filtered
and incubated for 10min at 90◦C. The sugars were purified
by semi-preparative HPLC using a system equipped with a
Waters Delta 600 pump and a 5?m Lichrosorb-NH2 column
(10mm×250mm) (An´ alisis V´ ınicos, Spain). An evaporative
light-scattering detector DDL-31 (Eurosep) and a fraction col-
lector (Waters) were used. The gradient method was closely
similar to that described above, but using a flow rate of
8.1mlmin−1. After collection of the different oligosaccharides,
water was eliminated by rotary evaporation.
2.7. Nuclear magnetic resonance (NMR)
The structure of the oligosaccharide was elucidated using a
HMQC) techniques. The spectra of the samples, dissolved
in deuterated water (ca. 10mM), were recorded on a Bruker
AVANCE 500 spectrometer equipped with a triple resonance
1H,13C,15N probe with a gradient in the Z axis, at a temper-
ature of 300K. Chemical shifts are in ppm with respect to the
0ppm point of DSS, used as internal standard. COSY, TOCSY
and NOESY experiments were performed with 16, 8 and 48
scans, respectively, with 256 increments in the indirect dimen-
sion and with 2048 points in the acquisition dimension. The
spectral widths were 9ppm in both dimensions. The HSQC
experiment (16 scans) also used 256 increments in the indirect
dimension and with 2048 points in the acquisition dimension.
The spectral width was 120ppm in the indirect dimension and
with a MALDI-TOF system Reflex III (Bruker-Franzen). For
the experiments the matrix employed was a saturated solution
of 2,5-hydroxybenzoic acid.
1H NMR (δ, ppm): 5.38 (H1G), 3.54 (H2G), 3.74 (H3G), 3.48
(H4G), 3.82 (H5G), 3.79 (H6aG), 3.76 (H6bG), 3.68 (H1aF1),
3.69 (H1bF1), 4.22 (H3F1), 4.06 (H4F1), 3.92 (H5F1), 3.95
(H6aF1), 3.82 (H6bF1), 3.77 (H1aF2), 3.65 (H1bF2), 4.16 (H3F2),
4.10 (H4F2), 3.88 (H5F2), 3.79 (H6aF2), 3.52 (H6bF2).13C
NMR (δ, ppm): 91.9 (C1G), 69.6 (C2G), 73.3 (C3G), 72.3
(C4G), 73.1 (C5G), 61.0 (C6G), 61.3 (C1F1), 76.4 (C3F1), 74.4
(C4F1), 81.6 (C5F1), 64.1 (C6F1), 61.5 (C1F2), 77.5 (C3F2), 74.5
(C4F2), 81.1 (C5F2), 63.5 (C6F2). Mass spectrometry: Calc. for
C18H32O16+Na=527.4. Found: [M+Na]+=527.1.
3. Results and discussion
3.1. Biochemical characterization of β-fructofuranosidase
3.1.1. Enzyme purification
Invertase is the major secreted protein when Sw. occidentalis
cells grow in lactose-based medium (Klein et al., 1989a). To
purify the enzyme, the culture supernatant of the yeast grown
on this carbon source was collected and processed as described
in Section 2. The enzyme was purified 300-fold to apparent
homogeneity, with an overall yield of 2% (data not shown). The
purified enzyme yields one 85kDa band on Coomassie-stained
SDS-PAGE (Fig. 1A). This mass is slightly higher than that
previously reported (76–78kDa) (Klein et al., 1989a). This dis-
crepancy could be the result of using different yeast strains and
mined by gel filtration on Sephadex G-200, and two active
forms of 165±11 and 85±7kDa (ratio 2:1) were obtained
(Fig. 1B). Surprisingly, however, only the smaller native form
was detected in gels under non-denaturing conditions (Fig. 1C).
Invertase from S. cerevisiae was used in a similar test as a con-
200kDa was detected (results not shown) corresponding to the
glycosylated, functionally active homodimer.
M.´Alvaro-Benito et al. / Journal of Biotechnology 132 (2007) 75–81
Fig. 1. SDS/PAGE analysis of the purified enzyme and molecular mass deter-
mination. (A) A Sw. occidentalis culture filtrate expressing invertase activity
before (lane 1) or after DEAE-Sephacel column chromatography pH 7 (lane 2)
by TCA and resuspended in 10?l of HCl–Tris 1.5M, pH 7.5. (B) Filtration in
Sephadex G-200 analysis. A280nm(open circles); ?-fructofuranosidase activ-
ity (closed circles) elution volumes were 66.5 and 77ml. (C) Purified Sw.
occidentalis invertase activity was revealed in situ (lane 1). The positions of
molecular mass markers (lane M) are indicated (in kDa) at the left and right of
(A and C).
Invertases from S. cerevisiae (Taussig and Carlson, 1983),
Schizosacch. pombe (Moreno et al., 1990) or P. anomala
(Rodr´ ıguez et al., 1995) are dimeric or multimeric enzymes,
with an average molecular weight of 60–65kDa for the non-
glycosylated-monomeric form. Two atypical yeast invertases
have also been described. One from A. adeninivorans has an
approximate molecular mass of 100kDa (Boer et al., 2004),
and another from R. glutinis has around 47kDa (Rubio et al.,
2002). The active forms of these two enzymes exist as a hex-
amer (600kDa) and a dimer (100kDa), respectively. In contrast
to most of the reported yeast invertases, the Sw. occidentalis
monomeric form (85kDa) described in this work shows ?-
fructofuranosidase activity (Fig. 1B). An active monomeric
invertase (60kDa) has also been described in C. utilis (Belcarz
et al., 2002) but this was a secreted non-glycosylated form,
which constituted only around 3% of the total invertase
Kinetic constants of ?-fructofuranosidase from Sw occidentalis on different
4.9 ± 1
1.3 ± 0.4
1.6 ± 0.8
1.3 ± 0.6
80.5 ± 6
26.3 ± 2
6.9 ± 1
10.9 ± 1
The ± refers to standard errors based on the curve fitting using SigmaPlot.
3.1.2. Substrate specificity and kinetic properties
The activity of the pure enzyme was examined with
sucrose as substrate at different temperatures and pHs in the
range 30–70◦C and 3–9U, respectively. It reached maxi-
mum values at pH 5.5 and 45–55◦C (results not shown).
The enzyme was able to liberate reducing sugars from
fructosyl-?(2→1)-linked carbohydrates such as sucrose [?-d-
glucopyranosyl-(1→2)-?-d-fructofuranose], 1-kestose [?-d-
fructofuranose] or raffinose [?-d-galactopyranosyl-(1→6)
-d-glucopyranosyl-(1→2)-?-d-fructofuranose]. The enzyme
displayed classical Michaelis–Menten kinetics towards all
these substrates (results not shown). Assuming that Kmreflects
the affinity of the enzyme for its substrate, the obtained values
suggest that Sw. occidentalis invertase has lower affinity
for sucrose than for the other tested fructooligosaccharides
(Table 1). The Km value of 4.9mM using sucrose is almost
three times higher than that found for the C. utilis enzyme
(1–2mM) (Belcarz et al., 2002), and lower than those mea-
sured for invertase from P. anomala (16mM) (Rodr´ ıguez
et al., 1995), S. cerevisiae (26.1mM) (Reddy and Maley,
1990) or A. adeninivorans (41mM) (Boer et al., 2004).
Nevertheless, the catalytic efficiency, defined by the ratio
kcat/Km, shows that the Sw. occidentalis enzyme hydrolyses
1-kestose or sucrose more efficiently than raffinose or nystose.
In addition, the enzyme had no activity on other glycosidic
bonds, e.g. those present in maltose [?-d-glucopyranosyl-
(1→4)-d-glucopyranose] or lactose [?-d-galactopyranosyl-
isomerssuch as turanose
(1→5)-d-fructofuranose] or palatinose [?-d-glucopyranosyl-
(1→6)-d-fructofuranose]. Most of these substrates were also
rans enzyme does show (low) hydrolytic activity for maltose,
turanose and lactose (Boer et al., 2004). This large size enzyme
has an amino acid sequence that shares a high degree of homol-
ogy with ?-glycosidases classified within family 31 of GH.
Clearly, the elucidation of the structure of the enzyme from Sw.
occidentalis, and its relation with substrate recognition, will be
as well ason sucrose
M.´Alvaro-Benito et al. / Journal of Biotechnology 132 (2007) 75–81
Fig. 2. (A) HPLC chromatogram corresponding to the reaction of sucrose with
the ?-fructofuranosidase from Sw. occidentalis. (1) Fructose; (2) glucose; (3)
sucrose; (4) 1-kestose; (5) 6-kestose. (B) Schematic view of the transfructosy-
3.2. Transfructosylating activity
The Sw. occidentalis ?-fructofuranosidase was assayed with
600gl−1sucrose using the conditions indicated in Section 2.
Analysis of the reaction products showed that the enzyme pre-
sented transfructosylating activity (Fig. 2A, peaks 4 and 5).
The amount of fructose detected was slightly smaller than
that detected for glucose, which is indicative of the fructosyl-
ing no peaks 4 and 5 (results not shown).
Based on its chromatographic mobility, the compound cor-
responding to peak 4 was identified as 1-kestose. The major
fructooligosaccharide in the reaction mixture (Fig. 2A, peak 5)
was purified by semi-preparative HPLC and its structure anal-
ysed by NMR. The1H NMR spectrum displayed one anomeric
signal corresponding to the glucose moiety. From this signal,
the combination of COSY, TOCSY, NOESY and HSQC allows
the deduction that all the1H and13C resonances relate solely
to the glucose residue. The NOESY cross peaks from glucose
H−1also allows us to identify the contiguous fructose moiety.
Again, the signals were resolved enough to enable the combi-
nation of COSY, TOCSY, NOESY and HSQC experiments to
identify the resonance signals of both this moiety and a second
fructose residue. Indeed, the NOESY experiment allows us to
conclude that the second fructose residue is ?-(2→6)-linked
to the fructose attached to the glucose moiety, thus unambigu-
ously identifying the compound as 6-kestose. Values obtained
for 6-kestose correlated well with those reported by Liu et al.
(1991). A schematic view of the biosynthetic reactions is shown
in Fig. 2B.
This trisaccharide exhibits enhanced prebiotic properties
compared with those of commercial FOS (Marx et al., 2000).
Such compounds exert beneficial effects on human health
as they are selectively fermented by colonic flora (Fukaya
et al., 1999). The enzymatic synthesis of 6-kestose and
other ?-(2→6)-linked fructosyl oligomers has been previously
reported. Straathof et al. (1986) suggested that the invertase
from S. cerevisiae formed 6-kestose at high sucrose concen-
trations (2.34M, 800gl−1), and using the same enzyme, Farine
et al. (2001) observed the release of d-glucose and d-fructose
and also identified five intermediate fructans, namely the dis-
accharides inulobiose and 6-?-fructofuranosylglucose, and the
trisaccharides 1-kestose, 6-kestose and neokestose. Chromato-
graphic studies also suggested that ?-fructofuranosidase from
Thermoascus aurantiacus synthesized 6-kestose (Katapodis
and Christakopoulos, 2004), and this oligosaccharide was also
poorly produced by the thermophilic fungus Sporotrichum ther-
mophile (Katapodis et al., 2004).
The maximal FOS production for a particular enzyme
depends on the relative rates of transfructosylation and hydrol-
ysis (Nguyen et al., 2005; Ballesteros et al., 2006). The time
course for the reaction of Sw. occidentalis ?-fructofuranosidase
is depicted in Fig. 3. The maximum concentration of 6-kestose
(76gl−1) was reached in 24h; at this time the total sucrose
conversion was close to 64%. At this point, the amount of 1-
kestose was approximately 25gl−1. This means a maximum
FOS production of 101gl−1, which corresponds to a 16.9%
(w/w) or an 8.4% (mol/mol) of the total carbohydrates in the
reaction mixture. The other sugars were glucose (25%, w/w),
fructose (22.4%, w/w) and sucrose (35.7%, w/w). After 24h,
kestoses were progressively hydrolysed (Fig. 3A), which is
observed in similar kinetically controlled glycosidase-catalysed
transformations. After 75h, the yield of 6-kestose stabilized in
The 6-kestose yield at the kinetic maximum obtained in
this work (Fig. 3) is, as far as we know, the highest yet
reported. The invertase from S. cerevisiae also produces 6-
kestose (Straathof et al., 1986; Farine et al., 2001), but
although the yield reported at the kinetic maximum was
only about 10% lower than that obtained using the Sw.
occidentalis enzyme, the fructooligosaccharides synthesized
were quickly hydrolysed. Thus, from a practical point of
view, the Sw. occidentalis ?-fructofuranosidase allows an
easier control of the 6-kestose production at the kinetic
M.´Alvaro-Benito et al. / Journal of Biotechnology 132 (2007) 75–81
?-fructofuranosidase from Sw. occidentalis. (B) Formation of total FOS.
Currently the main industrial FOS producer is the fructo-
syltransferase from Aspergillus (Sangeetha et al., 2005; Ghazi
et al., 2007). This enzyme provides a mixture of FOS of
the insulin-type structure ?-(2→1), without production of 6-
kestose. Several enzymes classified in family 32 of GH have
been reported in this organism, but none of them produces FOS
with the levan-type structure ?-(2→6). Accordingly, the Sw.
occidentalis enzyme as a 6-kestose (FOS) producer could pro-
vide a useful tool for biotechnology studies (Fern´ andez-Lobato
et al., 2005), but further protein structural analysis will be nec-
essary to fully understand the enzyme’s biological function and
to improve the potential of this enzyme.
of Education and Science (BIO2004-03773-C04), by Genoma
Espa˜ na, the National Foundation for Promoting Genomics and
Proteomics, and by an institutional grant from the Fundaci´ on
Ram´ on Areces to the Centro de Biolog´ ıa Molecular Severo
Ochoa. We thank Dr. Michael Cannon, King’s College, Lon-
don, for critically reading and correcting this paper. M.A. was
supported by a Spanish FPU fellowship from the Ministerio de
Educaci´ on y Ciencia.
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