Quantitative analysis of phytate globoids isolated from wheat bran and characterization of their sequential dephosphorylation by wheat phytase

Article abstract of DOI:10.1021/jf071191t

Wheat phytase was purified to investigate the action of the enzyme toward its pure substrate (phytic acid - myo-inositol hexakisphosphate) and its naturally occurring substrate (phytate globoids). Phytate globoids were purified to homogeneity from wheat bran, and their nutritionally relevant parameters were quantified by ICP-MS. The main components of the globoids were phytic acid (40% w/w), protein (46% w/w), and several minerals, in particular, K > Mg > Ca > Fe (in concentration order). Investigation of enzyme kinetics revealed that K_m and V_max decreased by 29 and 37%, respectively, when pure phytic acid was replaced with phytate globoids as substrate. Time course degradation of phytic acid or phytate globoids using purified wheat phytase was followed by HPIC identification of inositol phosphates appearing and disappearing as products. In both cases, enzymatic degradation initiated at both the 3- and 6-positions of phytic acid and end products were inositol and phosphate.

Full text of DOI:10.1021/jf071191t

J. Agric. Food Chem. 2007, 55, 7547−7552 7547
Quantitative Analysis of Phytate Globoids Isolated from Wheat
Bran and Characterization of Their Sequential
Dephosphorylation by Wheat Phytase
LISBETH BOHN,† LONE JOSEFSEN,† ANNE S. MEYER,§ AND
SØREN K. RASMUSSEN*,†
Department of Agricultural Sciences, Faculty of Life Sciences, University of Copenhagen,
Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark, and Bioprocess Engineering, Department of
Chemical Engineering, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
Wheat phytase was purified to investigate the action of the enzyme toward its pure substrate (phytic
acid - myo-inositol hexakisphosphate) and its naturally occurring substrate (phytate globoids). Phytate
globoids were purified to homogeneity from wheat bran, and their nutritionally relevant parameters
were quantified by ICP-MS. The main components of the globoids were phytic acid (40% w/w), protein
(46% w/w), and several minerals, in particular, K > Mg > Ca > Fe (in concentration order).
Investigation of enzyme kinetics revealed that Km and Vmax decreased by 29 and 37%, respectively,
when pure phytic acid was replaced with phytate globoids as substrate. Time course degradation of
phytic acid or phytate globoids using purified wheat phytase was followed by HPIC identification of
inositol phosphates appearing and disappearing as products. In both cases, enzymatic degradation
initiated at both the 3- and 6-positions of phytic acid and end products were inositol and phosphate.
KEYWORDS: Inositol phosphates; wheat phytase; wheat bran; phytate globoids; minerals; kinetics;
Triticum aestivum
INTRODUCTION
organized in clusters (6). In rice it has been shown that 11% of
the globoids consist of proteins with Mg and K in large amounts
(7). In wheat, the greatest concentrations of minerals have been
found in the bran (8, 9). Direct positive associations between
grain phytic acid and Zn have been reported (10), and it has
been shown that redistribution within the kernel of phosphate
and Mg, from the aleurone layer to the flour, takes place in the
wheat mutant Js-12-LPA (11).
Phytic acid is the main phosphorus storage compound in most
seeds and grains. The phytic acid molecule consists of a
phosphorylated myo-inositol ring and has a very specific
conformation with one axial and five equatorial phosphate
groups. Phytic acid is a strong chelating agent. It readily binds
metal cations from, for example, Ca, Fe, Zn, Mg, and Mn,
making them insoluble and thus unavailable as nutritional factors
(1). The salt of phytic acid and these minerals is known as
phytate, and it is concentrated in electron-dense parts of the
protein storage vacuoles called phytate globoids (2). How this
interaction between the protein storage vacuole and the phytate
globoid exactly is assembled is still being investigated. It could
be that either the proteins form a matrix with a few large phytate
globoids inside or there could actually be an inner membrane
in the vacuole surrounding the globoid (3). The globoids are
localized predominantly in the protein storage vacuoles in the
aleurone layer (wheat and barley) or in the embryo (maize) (4).
The size of the phytate globoids depends on the amount of phytic
acid in the grain. In wild-type wheat, globoids up to 4 µm in
diameter have been detected (5), whereas a low phytic acid
wheat mutant (Js-12-LPA) with the same amount of phosphate
but a lower content of phytic acid has smaller globoids,
Phytic acid can be degraded by phytase enzymes (myo-inositol
hexakisphosphate phosphohydrolase, EC 3.1.3.26), which are
defined as a class of phosphatases with the in vitro capability
to release at least one phosphate group from phytic acid, thereby
releasing the minerals and phosphorus. Wheat phytase was first
mentioned by Posternak and Posternak in 1929 (12), but it was
not until 1973 that Lim et al. concluded that there were two
enzymes in wheat, Phy1 and Phy2, with the ability to hydrolyze
phytic acid (13). Phy1 has a pH optimum of 6.0 and an optimal
temperature of 45 °C, whereas Phy2’s optimum pH lies at 5.5
with an optimal temperature of 50 °C. Vmax for Phy1 is about
half the Vmax of Phy2, and Phy1 is more easily inhibited by Pi
than Phy2 (14). One wheat phytase cDNA has been cloned,
and the sequence revealed that the enzyme has similarity to the
class of purple acid phosphatases (15). The hydrolysis of phytic
acid by wheat phytase has been shown to be inhibited when
the phytate is in complex with the minerals Al3+, Cu2+, Fe2+
,
* Author to whom correspondence should be addressed [e-mail
skr@life.ku.dk; telephone (+ 45) 35333436; fax (+45) 35283460].
† University of Copenhagen.
Fe3+, and Zn2+ (16). Phytic acid degradation by wheat phytase
§ Technical University of Denmark.
has been investigated on a number of occasions in connection
10.1021/jf071191t CCC: $37.00
© 2007 American Chemical Society
Published on Web 08/15/2007

7548 J. Agric. Food Chem., Vol. 55, No. 18, 2007
Bohn et al.
Figure 1. Hanes plot of wheat phytase kinetics using phytic acid as
Figure 3. Hanes plot of wheat phytase kinetics using phytate globoids
substrate at pH 6.0 and 45
the plot, Km is calculated to 0.83 mM phytic acid and Vmax to 0.230 mmol
of Pi/min mg.
°C. Error bars are indicated with lines. From
as substrate at pH 6.0 and 45
From the plot, Km is calculated to 0.96 mg of globoids/mL and Vmax to
0.185 mmol of Pi/min mg.
°C. Error bars are indicated with lines.
‚
‚
Phytase Purification. Chromatography was done on a fast protein
liquid chromatography system (A¨ ktaExplorer10S) from Amersham
Biosciences (Uppsala, Sweden). Wheat phytase was purified by gel
filtration on a HiPrep 26/60 Sephacryl S-200 column (Amersham
Biosciences). NaAc (0.1 M) containing 0.15 M NaCl, pH 5.5, was used
as running buffer. Crude phytase extract (250 mg) was dissolved in 2
mL of running buffer and filtered through a 0.22 µm filter before
injection. Fractions (1.8 mL) were tested for phytase activity as
described below, and positive samples were pooled and concentrated
using Millipore’s Amicon Ultra-4 10000 MWCO filter device (Carrigt-
wahill, Ireland). Purity was verified by SDS-PAGE and Coomassie blue
staining (Pierce GelCode 24590). The Bradford reagent was used for
protein quantification throughout the purification steps using BSA as
reference.
Enzyme Assay. Phytase activity was measured according to the
method given in ref 20 (adapted for Eppendorf tubes) at 42 °C in a
buffer containing 200 mM sodium acetate, pH 5.5, and 1 mM CaCl2.
The reaction was started by adding 200 µL of preheated substrate (7.5
mM phytic acid, 7.5 mM p-nitrophenyl phosphate, or 7.5 mM
1-naphthyl-phosphate), pH 5.5, to the enzyme (100 µL) and incubated
for 60 min. The reaction was terminated by adding 200 µL of color
stop mix (2.5% ammonium heptamolybdate, 5 mM ammonium vana-
date, and 10.7% nitric acid). Released phosphate was recorded by
measuring absorption spectrophotometrically at 415 nm using FLUOstar
galaxy (BMG labtech, Offenburg, Germany) and quantified using a
standard curve in the range of 0-500 nmol of NaH2PO4 dissolved in
the assay buffer. The activity of phytase was calculated as release of
inorganic phosphate (Pi) per minute.
Enzyme Characterization. Activity response to pH was examined
at 42 °C using a 0.1 M glycine buffer in the pH ranges of 2-4 and
9-10, 0.1 M sodium acetate buffer in the pH range of 4-6.5, and a
0.1 M Tris buffer in the pH range of 6-9. Measurements were done
in triplicate using the enzyme assay described above. The thermosta-
bility experiments were performed on a heating block (Grant QBTB,
Grant Instrument). The optimum temperature was determined in 200
mM sodium acetate, 1 mM CaCl2 at pH 5.5 and 7.5 mM phytic acid as
substrate in the range from 25 to 60 °C with 5 °C increments. Samples
were equilibrated at target temperature for 15 min before the enzyme
was added and then incubated for an additional 60 min. The reaction
was stopped by adding color stop mix. Km and Vmax measurements were
performed under optimal conditions (45 °C and pH 6.0) using various
concentrations of either phytate globoids or phytic acid as substrate to
0.28 mg enzyme and incubated for 30 minutes.
Figure 2. Phytate globoids isolated from wheat bran. Globoids are the
spheres visualized by light microscopy ( 100). Scale bar 20 m.
×
)
µ
with characterizations of the enzyme (17, 18), and it has been
determined that it acts through attack on phosphate in the
6-position to yield myo-inositol as the final product.
The degradation patterns documented so far were, however,
based on experiments in which soluble phytic acid was present
for degradation. It has been proved that soluble phytic acid can
be completely degraded under these conditions, but not whethers
and howsphytase degrades the naturally occurring form of
phytic acidsthe insoluble phytate globoids. The importance of
a proper degradation of phytate to improve mineral bioavail-
ability from food has recently been extensively reviewed (19).
The inositol phosphates must be reduced to very low levels to
prevent inhibition of mineral bioavailability, and the understand-
ing of the natural action of endogenous plant phytases on phytate
globoids is of economic importance to the food-processing
industry.
This study was undertaken to investigate the relationships
between wheat phytase, phytic acid, and phytate globoids. The
isolated phytate globoids are characterized with respect to
mineral composition as compared with the bran fraction.
Furthermore, the degradation patterns by wheat phytase of pure
phytic acid and phytate globoids isolated from wheat are
established and the kinetics of phytase using either phytic acid
or phytate globoids are compared.
Isolation of Phytate Globoids. Aleurone particles were purified from
wheat bran following the nonaqueous protocol of Tanaka et al. (7)
through six centrifugation steps. Sixty grams of bran was homogenized
with 100 mL of cottonseed oil in a Braun blender (MX32, Frankfurt,
Germany) at maximum speed for 10 min at room temperature. After
the homogenate had been filtered through gauze, the slurry was
centrifuged at 2600g for 15 min and the supernatant was discharged.
The pellet was suspended in cottonseed oil/CCl4 mixtures, which were
carried through the subsequent steps as outlined by Tanaka et al. (7).
Quantifications of Phytate Globoid Components. Two hundred
milligrams of material was treated as described in ref 21 before the
MATERIALS AND METHODS
Plant Material, Chemicals, and Enzymes. Wheat bran was kindly
provided by Ringsted Dampmølle, Ringsted, Denmark. Phytic acid
isolated from corn (P8810), crude extract of wheat phytase (P1255),
bovine serum albumin (BSA) (A7906), cotton seed oil (C7767),
p-nitrophenyl phosphate (104-0), and the Bradford reagent (B6916) were
all purchased from Sigma-Aldrich (St. Louis, MO). Anhydrous carbon
tetrachloride (CCl4) was purchased from Prolabo (Fontenay sous Bois,
France), and 1-naphthylphosphate sodium salt monohydrate (6815) was
purchased from Merck (Darmstadt, Germany).

Wheat Bran Phytate Globoids
J. Agric. Food Chem., Vol. 55, No. 18, 2007 7549
Figure 4. Chromatographic profile of (bottom) hydrolyzed sodium phytate and partly degraded phytate globoids after 0 (center) and 15 min incubation
(top). Peaks: 3, InsP2; 4, InsP2; 5 and 6, unidentified; 7, DL-Ins(1,2,4)P3, DL-Ins(1,3,4)P3, and Ins(1,2,3)P3; 8, DL-Ins(1,2,6)P3 and Ins(1,2,3)P3; 9,
1−
DL-Ins(1,4,5)P3; 10, DL-Ins(1,5,6)P3; 11, DL-Ins(4,5,6)P3; 12, Ins(1,2,3,5)P4; 13, DL-Ins(1,2,4,6)P4; 14, DL-Ins(1,2,3,4)P4; 15, Ins(1,3,4,6)P4; 16, DL-Ins-
(1,2,4,5)P4; 17, DL-Ins(1,3,4,5)P4; 18, DL-Ins(1,2,5,6)P4; 19, Ins-(2,4,5,6)P4; 20, DL-Ins(1,4,5,6)P4; 21, Ins(1,2,3,4,6)P5; 22, DL-Ins(1,2,3,4,5)P5; 23, DL-Ins-
(1,2,4,5,6)P5; 24, Ins(1,3,4,5,6)P5; 25, InsP6.
Figure 5. Wheat phytase degradation of phytate globoids from wheat. The peaks are not quantified, but the time of the maximum height tells us in which
order the products are produced. A similar experiment was performed using pure phytic acid as substrate, and the pattern was the same, although the
reaction was faster (data not shown).
Table 1. Quantification of the Composition of Major Elements and Minerals in Wheat Phytate Globoids Compared to Wheat Bran (Average
Coefficient of Variation <4 %)
mg/g
µg/g
phytic acid
protein
moisture
B
Ca
Cu
Fe
K
Mg
Mn
Na
P
S
Zn
globoid
bran
402
129
462
13
103
73
1.3
2.0
4290.1
832.9
58.0
11.3
589.0
82.6
76443.9
17157.6
32351.4
4377.5
122.2
71.7
318.1
79.9
81739.8
11616.4
1724.1
1227.8
223.1
47.4
composition of the minerals Al, B, Ca, Cu, Fe, K, Mg, Mn, Mo, Na,
Ni, P, S, and Zn was measured by inductively coupled plasma-mass
spectrometry (ICP-MS) (Agilent 7500ce, Agilent Technologies, Manches-
ter, U.K.). Proteins were extracted in Milli-Q water overnight at 8 °C
and quantified using the Bradford reagent. Phytic acid was extracted
in 0.1 M HCl overnight at 8 °C and quantified on HPIC as described
below. Moisture was removed by freeze-drying samples overnight
(Alpha 1-4, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode
am Harz, Germany). The potential presence of starch in the globoids
was assessed by quantitative measurement of glucose by high-
performance anionic chromatography (HPAEC) of globoid hydrolysates
resulting after acid hydrolysis (2 M HCl, 100 °C, 1 h). ICP-MS
quantifications were done in triplicates. Protein, phytic acid, and
moisture quantifications were done 5-fold.
Hydrolysis of Phytic Acid by Phytase. Six milligrams of phytate
or phytate globoids was incubated in 2.3 mL of 200 mM sodium acetate,
pH 5.5, and 1 mM CaCl2 buffer. The assay was started by adding 3.3
units of preheated phytase in 0.2 mL of buffer to the reaction mixture.

7550 J. Agric. Food Chem., Vol. 55, No. 18, 2007
Bohn et al.
Figure 6. Degradation pattern of phytic acid in globoids by purified Sigma wheat phytase as suggested from the data of Figure 5. In this figure only,
P represents inorganic phosphate.
At appropriate times from -2 min to 1260 min 100 µL was transferred
to 200 µL of 95 °C Milli-Q water, and the enzyme was inactivated by
incubation at 95 °C for 15 min. Samples were spun at full speed on an
Eppendorf Centrifuge 5417R (Radiometer, Brønshøj, Denmark) for 2
min before the degradation products were extracted with 0.4 M HCl,
filtered, and analyzed at HPIC.
the differences observed using the pure phytic acid or phytate
globoids as substrate for the measurements under exactly the
same conditions, as we describe in the following sections.
Phytate Globoid Purification and Characterization. Par-
ticles from the aleurone layer were isolated from wheat bran
by a six-step differential centrifugation procedure using non-
aqueous media. After the final purification step, the preparation
had an ivory white powder appearance and was only partially
soluble in aqueous solutions. The particles were spherical with
diameters ranging from ∼1.5 to 5 µm (Figure 2). This is
comparable to the average size globoid of 4 µm for aleurone
grains in wheat bran (5). The yield of globoids was in the range
of 0.4-0.7% (w/w) of the initial weight of the bran. These
phytate globoids contained 402 mg of phytic acid/g of globoid,
462 mg of protein/g of globoid, 10.3% moisture, high contents
of P (82 mg/g) and the minerals Mg and K (32-76 mg/g), and
relatively high contents of Fe and Ca (0.6-4.3 mg/kg) (Table
1). Besides the listed minerals Al, Mo, and Ni were also
quantified, but with too high variation to make the values
significant. No starch granules were observed visually by light
microscope (Figure 2), nor was any glucose detected in the
particles by HPAEC after acid hydrolysis. We will therefore
from now on refer to them as phytate globoids. In general, an
increase in the concentration of several cations during the
purification of the globoids was observed (Table 1). The
concentrations of Fe, Mg, and P were all 7-fold higher in the
purified globoids as compared with wheat bran. This copurifi-
cation of Fe and Mg with phytate-P allows us to suggest an
association between these compounds and confirms a connection
between phytic acid and Mg, verifying what has been docu-
mented in low-phytate wheat [Js-12-LPA (11)] and rice [lpa1-
1 (24)]. Other minerals such as Ca, Zn, and K increased 5-fold
in concentration from wheat bran to purified globoids (Table
1). This indicates that they are more loosely bound to the phytic
acid, and it can be inferred that they also are stored in other
parts of the seed or bran. This theory is supported by the fact
that compared with whole wheat grain (25, 26), the concentra-
tions of Ca and Fe in bran are both about twice as high in bran
compared to the whole grain, but in the enrichment step from
bran to globoids, the relative increase in Fe is much higher than
what is detected for Ca.
Quantification of Inositol Phosphates. Phytic acid and other inositol
phosphates were quantified on a HPLC (chemically inert HPLC system
10Avp series, Shimadzu, Kyoto, Japan) equipped with a guard column
(Dionex CarboFac PA-100, 4 × 50 mm) and a HPIC CarboPac PA-
100 (4 × 250 mm) analytical column (Dionex Corp., Sunnyvale, CA)
at 40 °C in a column oven (CTO-10A). The protocol was performed
according to that of Carlsson et al. (22). Samples (100 µL) were loaded
with an autoinjector (SIL-10Ai). The eluants (0.8 mL/min) were mixed
with 0.1% Fe(NO3)3‚9H2O (Sigma-Aldrich) in a 2% solution of HClO4
in a postcolumn reactor using a Shimadzu HPLC pump LC-10Ai. The
combined flow rate was 1.2 mL/min. A mixing tee and a 4 m PTFE
knitted open tubular reactor coil 1/16 in. o.d., 0.25 mm i.d. (from a local
supplier) was used to ensure sufficient reaction time and mixing. Phytate
was detected after postcolumn reaction by monitoring the absorbance
at 290 nm using a Shimadzu SPD-M10Avp diode array detector. A
hydrolysate of phytic acid was prepared by boiling pure phytic acid
for 12 h in 6 M HCl, vaporizing the acid, and redissolving the sample
in Milli-Q water. The peaks were then compared with the hydrolysate
of refs 22 and 23. For data analysis Class-VP, release 6.12 SP
(Shimadzu) was used.
Statistical Analysis. Data are presented as means ( standard
deviation (n ) 3). In Figures 1 and 3 linear regression is used and
linearity (R2) is calculated using Microsoft Excel 2003.
RESULTS AND DISCUSSION
Phytase Purification and Characterization. Size exclusion
chromatography of the crude extract of wheat phytase resulted
in one peak of activity, and the fractions containing the activity
were pooled and concentrated; subsequently, a 4-fold increase
in activity was shown as compared to the activity of the crude
phytase extract. Purity was tested by SDS-PAGE of the fractions
collected after chromatography. It revealed copurification of two
proteins of almost identical size, of which only one had phytase
activity. The phytase displayed highest activity at pH 6.0 and
an optimum temperature of 45 °C (data not shown), which is
in agreement with previously published results for wheat Phy1
(14). The phytase was also inhibited by 0.3 mM sodium
phosphate added to the solution as expected from previous
descriptions of Phy1 and its sensitivity to product inhibition
(13, 14). When pure sodium salt of phytate was used as substrate
(Figure 1), Km (0.83 mM phytic acid) was higher than
previously reported values for Phy1, which range from 0.02
mM (13) to 0.48 µM (14). Vmax (0.230 mmol of Pi/min‚mg of
protein) corresponded well with the Vmax value of 127 µmol/
min‚mg of protein for purified wheat phytase previously reported
by Nakano et al., considering differences in buffer systems and
assay conditions (14). More comparable than these values were
Degradation of Phytate Globoids by Phytase. Because
phytate in globoids may be embedded in proteins (2), it is not
expected to be as readily accessible to dephosphorylation as
chemically pure phytic acid. We have tested the way that phytase
behaves toward its natural substrate (Figure 3). When phytate
globoids were used as substrate, Km and Vmax were calculated
to 0.96 mg of globoids/mL [equivalent to 0.59 mM phytic acid/
mg of protein based on the levels of phytate determined in the
globoids (Table 1)] and 0.185 mmol of Pi/min‚mg of protein,

Wheat Bran Phytate Globoids
J. Agric. Food Chem., Vol. 55, No. 18, 2007 7551
respectively. Both of these values are significantly lower than
the values computed for wheat phytase when phytic acid was
the substrate (Figure 1). Km using phytate globoids decreased
by 29% as compared to the value determined using chemically
pure phytate as substrate, and Vmax decreased 37% under similar
conditions. This result could be explained as a simple question
of space: The proteins in the globoid may retard the action of
the enzyme simply by blocking access to phytic acid. If phytate
crystals are embedded in a protein matrix or membrane (2), the
enzymatic activity may be hindered by the structure of the
complex. A similar experiment using the smaller globoids from
a low phytic acid mutant (24) could investigate this theory,
because a small membrane-covered phytate crystal would be
expected to have the same Km and Vmax values as larger globoids,
if the proteins covered that globoid with a membrane. An
alternative explanation could be that the minerals in complex
with the phytic acid inhibit dephosphorylation by forming
insoluble salts as studied by Tang et al. (16). However, in this
investigation HPIC analysis of phytic acid and its lower inositol
phosphate products (Figures 4-6) showed that wheat phytase
was able to attack phytic acid in globoids and that all inositol
phosphates were completely degraded within 20 h (data not
shown). In addition, the speed of degradation slowed, especially
in the last dephosphorylation steps from inositol di-kisphosphate
to inositol (Figure 5). This could be a consequence of product
inhibition, as the phosphate concentration slowly increased
during the reaction.
Manufacturers of food or feed with phytase added should
consequently expect a lower activity level and a lower yield of
phosphate and free minerals than the values calculated in
advance.
ABBREVIATIONS USED
HPIC, high-performance ion chromatography; InsPx, inositol
mono-hexakisphosphate; WT, wild type; PA, phytic acid; Pi,
inorganic phosphate.
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In conclusion, we have established a connection between
several minerals and phytate globoids. Furthermore, data
here shown indicated that wheat phytase apparently was
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Despite inhibition from other substances in the globoids, the
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the 6- or 3-positioned C in the inositol hexakisphosphate ring.
Finally, use of the naturally occurring phytate globoids as
substrate for phytase slowed the action of the enzyme compared
to using commercially available phytic acid as substrate.

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Received for review April 23, 2007. Revised manuscript received June
14, 2007. Accepted June 26, 2007. This study was financed by the Danish
Agency for Science, Technology and Innovation, Copenhagen, Denmark.
JF071191T